EP4142498A1 - Insecticidal combinations - Google Patents

Insecticidal combinations

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Publication number
EP4142498A1
EP4142498A1 EP21727053.7A EP21727053A EP4142498A1 EP 4142498 A1 EP4142498 A1 EP 4142498A1 EP 21727053 A EP21727053 A EP 21727053A EP 4142498 A1 EP4142498 A1 EP 4142498A1
Authority
EP
European Patent Office
Prior art keywords
toxin
photorhabdus
bacillus thuringiensis
combination
amino acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP21727053.7A
Other languages
German (de)
English (en)
French (fr)
Inventor
Kyle Schneider
Breck DAVIS
Joseph TOURTOIS
Daniel Hulbert
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Vestaron Corp
Original Assignee
Vestaron Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Vestaron Corp filed Critical Vestaron Corp
Publication of EP4142498A1 publication Critical patent/EP4142498A1/en
Pending legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/50—Isolated enzymes; Isolated proteins
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
    • A01N37/44—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a nitrogen atom attached to the same carbon skeleton by a single or double bond, this nitrogen atom not being a member of a derivative or of a thio analogue of a carboxylic group, e.g. amino-carboxylic acids
    • A01N37/46—N-acyl derivatives
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N59/00—Biocides, pest repellants or attractants, or plant growth regulators containing elements or inorganic compounds
    • A01N59/14—Boron; Compounds thereof
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/10—Animals; Substances produced thereby or obtained therefrom
    • A01N63/12—Nematodes
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
    • A01N63/22—Bacillus
    • A01N63/23—B. thuringiensis
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/30—Microbial fungi; Substances produced thereby or obtained therefrom
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/40—Viruses, e.g. bacteriophages
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
    • A01N65/08—Magnoliopsida [dicotyledons]
    • A01N65/26—Meliaceae [Chinaberry or Mahogany family], e.g. mahogany, langsat or neem
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
    • A01P7/00—Arthropodicides
    • A01P7/04—Insecticides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • TECHNICAL FIELD New insecticidal combinations of Cysteine Rich Insecticidal Proteins (CRIPs), and Insecticidal Agents (IAs), e.g., chemical substances, molecules, nucleotides, polynucleotides, peptides, polypeptides, proteins, toxins, toxicants, poisons, insecticides, pesticides, organic compounds, inorganic compounds, prokaryote organisms, or eukaryote organisms (and the agents produced from said prokaryote or eukaryote organisms), for the control and/or eradication of pests are described and claimed.
  • CRIPs Cysteine Rich Insecticidal Proteins
  • IAs Insecticidal Agents
  • Numerous insects are vectors for disease.
  • Mosquitoes in the genus Anopheles are the principle vectors of Zika virus, Chikungunya virus, and malaria, a disease caused by protozoa in the genus Trypanosoma.
  • Aedes aegypti is the main vector of the viruses that cause Yellow fever and Dengue.
  • Other viruses, the causal agents of various types of encephalitis, are also carried by Aedes spp. mosquitoes.
  • Wuchereria bancrofti and Brugia malayi parasitic roundworms that cause filariasis, are usually spread by mosquitoes in the genera Culex, Mansonia, and Anopheles.
  • Horse flies and deer flies may transmit the bacterial pathogens of tularemia (Pasteurella tularensis) and anthrax (Bacillus anthracis), as well as a parasitic roundworm (Loa loa) that causes loiasis in tropical Africa.
  • Eye gnats in the genus Hippelates can carry the spirochaete pathogen that causes yaws (Treponema per pneumonia), and may also spread conjunctivitis (pinkeye).
  • Tsetse flies in the genus Glossina transmit the protozoan pathogens that cause African sleeping sickness (Trypanosoma gambiense and T.
  • IAs are one or more chemical substances, molecules, nucleotides, polynucleotides, peptides, polypeptides, proteins, toxins, toxicants, poisons, insecticides, pesticides, organic compounds, inorganic compounds, prokaryote organisms and/or the products therefrom (such as bacterial toxins), or eukaryote organisms and/or the products therefrom (such as fungal toxins). IAs can be combined to provide insecticidal effects that are greater than the additive effect of any IA used in isolation.
  • CRIPs are peptides, polypeptides, and/or proteins that possess cysteine residues that, in some embodiments, are capable of forming disulfide bonds; these disulfide bonds create a scaffolding motif that is observed in a wide variety of unrelated protein families.
  • An example of peptides that fall within the CRIP family are inhibitor cystine knot (ICK) peptides.
  • ICK peptides include many molecules that have insecticidal activity. Such ICK peptides are often toxic to naturally occurring biological target species, usually insects or arachnids of some type. Often ICK peptides can have arthropod origins such as the venoms of scorpions or spiders.
  • IAs and CRIPs we describe novel insecticidal combinations of IAs and CRIPs.
  • an insecticidally effective combinations comprising (1) one or more CRIPs, or pharmaceutically acceptable salts thereof; one or more CRIP-insecticidal proteins, or pharmaceutically acceptable salts thereof; or a combination thereof; and (2) one or more Insecticidal Agents (IA), and methods of using the same to preserve the crops we depend on for food, and safeguard human and animal health.
  • IA Insecticidal Agents
  • This invention describes how to combine CRIPs and IAs so they provide insecticidal effects that are greater than the additive insecticidal effect of any IA or CRIP used in isolation.
  • the present disclosure describes how to make and use combinations of CRIPS and IAs to kill and control insects, even insecticide-resistant insects, and even at low doses. Without being bound by theory, our understanding of CRIPs and IAs allows us to teach one ordinarily skilled in the art, to create novel methods, compositions, compounds (proteins and peptides) and procedures to protect plants and control insects. [0012]
  • the present disclosure describes a combination comprising a Cysteine Rich Insecticidal Peptide (CRIP) and an Insecticidal Agent (IA).
  • CRIP Cysteine Rich Insecticidal Peptide
  • IA Insecticidal Agent
  • the present disclosure describes a combination comprising a Cysteine Rich Insecticidal Peptide (CRIP) and an Insecticidal Agent (IA), wherein the IA is a bacterial toxin; a fungal toxin; a lectin; an Azadirachta indica compound; a boron compound; a virus; or a combination thereof; and wherein the CRIP is a U1-agatoxin-Ta1b peptide; a U1-agatoxin-Ta1b Variant Polypeptide (TVP); a sea anemone toxin; an Av3 Variant Polypeptide (AVP); a Phoneutria toxin; or an Atracotoxin (ACTX).
  • CRIP Cysteine Rich Insecticidal Peptide
  • IA Insecticidal Agent
  • the present disclosure describes a composition comprising the combination a combination comprising a Cysteine Rich Insecticidal Peptide (CRIP) and an Insecticidal Agent (IA), and further comprising an excipient.
  • CRIP Cysteine Rich Insecticidal Peptide
  • IA Insecticidal Agent
  • the present disclosure describes a combination comprising one or more fermentation solids, spores, or toxins isolated from a Bacillus thuringiensis ssp. kurstaki strain EVB-113-19, and a U1-agatoxin-Ta1b peptide having an amino acid sequence according to the amino acid sequence set forth in SEQ ID NO: 1.
  • the present disclosure describes a combination comprising one or more fermentation solids, spores, or toxins isolated from a Bacillus thuringiensis ssp. kurstaki strain EVB-113-19, and a U1-agatoxin-Ta1b Variant Polypeptide (TVP) having an amino acid sequence according to the amino acid sequence set forth in SEQ ID NO: 2.
  • TVP U1-agatoxin-Ta1b Variant Polypeptide
  • the present disclosure describes a combination comprising one or more fermentation solids, spores, or toxins isolated from a Bacillus thuringiensis ssp.
  • the present disclosure describes a combination comprising one or more fermentation solids, spores, or toxins isolated from a Bacillus thuringiensis ssp. kurstaki strain EVB-113-19, and a ⁇ -CNTX-Pn1a toxin having an amino acid sequence according to the amino acid sequence set forth in SEQ ID NO: 65.
  • the present disclosure describes a combination comprising a Beauveria bassiana strain ANT-03 spore, and a U+2-ACTX-Hv1a toxin having an amino acid sequence according to the amino acid sequence set forth in SEQ ID NO: 61.
  • the present disclosure describes a combination comprising one or more fermentation solids, spores, or toxins isolated from a Bacillus thuringiensis ssp. tenebrionis strain NB-176, and a U+2-ACTX-Hv1a toxin having an amino acid sequence according to the amino acid sequence set forth in SEQ ID NO: 61.
  • the present disclosure describes a combination comprising one or more fermentation solids, spores, or toxins isolated from a Bacillus thuringiensis ssp. kurstaki strain EVB-113-19, and a U+2-ACTX-Hv1a toxin having an amino acid sequence according to the amino acid sequence set forth in SEQ ID NO: 61.
  • the present disclosure describes a combination comprising one or more fermentation solids, spores, or toxins isolated from a Bacillus thuringiensis ssp.
  • the present disclosure describes a combination comprising a Photorhabdus luminescens toxin, and an ACTX; wherein the Photorhabdus luminescens toxin is a Photorhabdus luminescens toxin complex (Tca) comprising a TcaA (SEQ ID NO: 616), a TcaB (SEQ ID NO: 617), a TcaC (SEQ ID NO: 618), and a TcaZ (SEQ ID NO: 619); and wherein the ACTX peptide is a U+2-ACTX-Hv1a toxin (SEQ ID NO: 61).
  • Tca Photorhabdus luminescens toxin complex
  • the ACTX peptide is a U+2-ACTX-Hv1a toxin (SEQ ID NO: 61).
  • the present disclosure describes a combination comprising a Galanthus nivalis agglutinin (GNA), and an ACTX; wherein the GNA has an amino acid sequence as set forth in SEQ ID NO: 35; and wherein the ACTX peptide is a U+2-ACTX- Hv1a toxin having an amino acid sequence according to the amino acid sequence set forth in SEQ ID NO: 61.
  • GNA Galanthus nivalis agglutinin
  • ACTX a U+2-ACTX- Hv1a toxin having an amino acid sequence according to the amino acid sequence set forth in SEQ ID NO: 61.
  • the present disclosure describes a combination comprising an Azadirachtin, and an ACTX; wherein the Azadirachtin is an Azadirachtin having a chemical formula: C 35 H 44 O 16 ; and wherein the ACTX is a U+2-ACTX-Hv1a toxin having an amino acid sequence according to the amino acid sequence set forth in SEQ ID NO: 61.
  • the present disclosure describes a combination comprising a boric acid compound, and an ACTX; wherein the boric acid compound has a chemical formula of H 3 BO 3 ; and wherein the ACTX peptide is a U+2-ACTX-Hv1a toxin having an amino acid sequence according to the amino acid sequence set forth in SEQ ID NO: 61.
  • the present disclosure describes a combination comprising a Cydia pomonella granulovirus (CpGV), and an ACTX; wherein the CpGV is a Cydia pomonella granulovirus isolate V22 virus; and wherein the ACTX peptide is a U+2-ACTX-Hv1a toxin having an amino acid sequence according to the amino acid sequence set forth in SEQ ID NO: 61.
  • CpGV Cydia pomonella granulovirus
  • ACTX peptide is a U+2-ACTX-Hv1a toxin having an amino acid sequence according to the amino acid sequence set forth in SEQ ID NO: 61.
  • the present disclosure describes a method of using a combination comprising a Cysteine Rich Insecticidal Peptide (CRIP) and an Insecticidal Agent (IA) to control insects, said method comprising, providing a combination of at least one CRIP and at least one IA, applying a combination comprising a Cysteine Rich Insecticidal Peptide (CRIP) and an Insecticidal Agent (IA)to the locus of an insect.
  • CRIP Cysteine Rich Insecticidal Peptide
  • IA Insecticidal Agent
  • the present disclosure describes a method of using a combination comprising a Cysteine Rich Insecticidal Peptide (CRIP) and an Insecticidal Agent (IA) to control Bacillus thuringiensis-toxin-resistant insects comprising, providing a combination of at least one CRIP and at least on IA; and then applying said combination to the locus of an insect.
  • CRIP Cysteine Rich Insecticidal Peptide
  • IA Insecticidal Agent
  • the present disclosure describes a method of combating, controlling, or inhibiting a pest comprising, applying a pesticidally effective amount of a combination comprising a Cysteine Rich Insecticidal Peptide (CRIP) and an Insecticidal Agent (IA) to the locus of the pest, or to a plant or animal susceptible to an attack by the pest.
  • CRIP Cysteine Rich Insecticidal Peptide
  • IA Insecticidal Agent
  • FIG.1 shows a graph depicting the 24-hour mortality of Aedes aegypti (mosquito) larvae after a diet incorporation assay using (1) U+2-ACTX-Hv1a with Bti; (2) Bti toxin alone; (3) U+2-ACTX-Hv1a alone; and (4) control (water).
  • FIG.2 depicts a graph showing the 3-day mortality of the Lepidopteran species, the beet armyworm (Spodoptera exigua) after a foliar spray assay with Bacillus thuringiensis var. kurstaki toxins (Btk) combined with ⁇ -CNTX-Pn1a.
  • FIG.3 depicts a graph showing the 3-day mortality of the Lepidopteran species, the beet armyworm (Spodoptera exigua), after a foliar spray assay with Btk combined with Av3-Variant Polypeptides (AVPs).
  • AVPs Av3-Variant Polypeptides
  • FIG.4 shows a chromatogram evaluating WT-Ta1b degradation in Helicoverpa zea gut extract (HGE), a simulated lepidopteran gut environment, after 0, 20, 40, 60, 180, and 1260 minutes. The box indicates the major and minor peaks, thus indicating the degradation of WT-Ta1b. Nested insets show zoomed in and zoomed out views of the chromatogram.
  • HGE Helicoverpa zea gut extract
  • FIG.5 shows a chromatogram evaluating TVP-R9Q degradation in Helicoverpa zea gut extract (HGE), a simulated lepidopteran gut environment, after 0, 20, 40, 60, 180, and 1260 minutes.
  • HGE Helicoverpa zea gut extract
  • the box indicates the presence of single peak, thus indicating the stability of TVP-R9Q.
  • Nested insets show zoomed in and zoomed out views of the chromatogram.
  • the presence of a single main peak indicates the stability of the TVP-R9Q peptide.
  • FIG.6 depicts a graph showing the results of a defoliation assay when testing WT-Ta1b, Btk toxins, and combinations thereof against the Lepidopteran species, Helicoverpa zea (corn earworm). Treatments were as follows: (1) WT-Ta1b alone; (2) Btk toxin alone; (3) a combination of both WT-Ta1b and Btk toxin; or (4) a control (0.125% Vintre, a surfactant).
  • FIG.7 depicts a graph showing the results of a defoliation assay when testing TVP-R9Q, Btk toxins, and combinations thereof against the Lepidopteran species, Helicoverpa zea (corn earworm). Treatments were as follows: (1) TVP-R9Q alone; (2) Btk toxin alone; (3) a combination of both TVP-R9Q and Btk toxin; or (4) a control (0.125% Vintre, a surfactant).
  • FIG.8 depicts a graph showing the results of a mortality assay when testing WT-Ta1b, Btk toxins, and combinations thereof against the Lepidopteran species, Helicoverpa zea (corn earworm). Treatments were as follows: (1) WT-Ta1b alone; (2) Btk toxin alone; (3) a combination of both WT-Ta1b and Btk toxin; or (4) a control (0.125% Vintre, a surfactant).
  • FIG.9 depicts a graph showing the results of a mortality assay when testing TVP-R9Q, Btk toxins, and combinations thereof against the Lepidopteran species, Helicoverpa zea (corn earworm). Treatments were as follows: (1) TVP-R9Q alone; (2) Btk toxin alone; (3) a combination of both TVP-R9Q and Btk toxin; or (4) a control (0.125% Vintre, a surfactant).
  • FIG.10 depicts a graph showing the 4-day mortality of the Coleopteran species, the Darkling Beetle (Alphitobius diaperinus) after a diet incorporation assay with (1) U+2-ACTX-Hv1a alone; (2) Btt toxin alone; (3) a combination of both U+2-ACTX-Hv1a and Btt toxin; or (4) an untreated control (water).
  • FIG.11 depicts a graph showing the 4-day mortality of the Colorado potato beetle (Leptinotarsa decemlineata) when sprayed with (1) U+2-ACTX-Hv1a alone; (2) Btt toxin alone; (3) a combination of both U+2-ACTX-Hv1a and Btt toxin; or (4) an untreated control (water).
  • FIG.12 depicts a graph showing mortality at day 4 in corn earworm larvae treated with: (a) Water; (b) Photorhabdus luminescens toxin complex extract alone (4.75% v/v); (c) 10 mg/mL U+2-ACTX-Hv1a (1% w/v); and (d) Photorhabdus luminescens toxin complex extract (4.75% w/v) with 10 mg/mL U+2-ACTX-Hv1a (1% w/v).
  • % w/v is percent w/v of the total volume of the composition, with the remainder being water.
  • FIG.13 depicts a graph showing the mortality rate in corn earworm neonates on day three after being treated with (a) 0 mg/mL GNA (0% w/v); with 0 mg/mL U+2- ACTX-Hv1a (0% w/v) (control); (b) 2.5 mg/mL GNA (0.25% w/v); 0 mg/mL U+2-ACTX- Hv1a (0% w/v); (c) 0 mg/mL GNA (0% w/v); 5 mg/mL U+2-ACTX-Hv1a (0.5% w/v); and (d) 2.5 mg/mL GNA (0.25% w/v); 5 mg/mL U+2-ACTX-Hv1a (0.5% w/v).
  • % w/v is percent w/v of the total volume of the composition, with the remainder being water.
  • Proportional mortality refers to the proportion of individual insects killed over the course of an experiment, i.e., the number of dead individuals over the total number of individuals.
  • FIG.14 depicts a graph showing the mortality rate in Fall armyworm (Spodoptera frugiperda) larvae on day three after being treated with (a) Chitinase 0 ⁇ L/L (0% w/v); U+2-ACTX-Hv1a 0 mg/mL (0% w/v); Sucrose (10% w/v); (b) Chitinase 100 ⁇ L/L (0.01% w/v); U+2-ACTX-Hv1a 0 mg/mL (0% w/v); Sucrose (10% w/v); (c) Chitinase 0 ⁇ L/L (0% w/v); U+2-ACTX-Hv1a 5 mg/mL (0.5% w/v); Sucrose (10% w/v); and (d) Chitinase 100 ⁇ L/L (0.01% w/v); U+2-ACTX-Hv1a 5 mg/mL (0.5% w/v);
  • FIG.15 depicts the chemical structure of the insect growth regulator, Azadirachtin.
  • FIG.16 depicts a graph showing the mortality rate in Corn earworm (Helicoverpa zea) neonates on day three after being treated with (a) 0 ⁇ L/L Azadirachtin (0% v/v); 0 mg/mL U+2-ACTX-Hv1a (0% w/v) (control); (b) 80 ⁇ L/L Azadirachtin (0.008% v/v); 0 mg/mL U+2-ACTX-Hv1a (0% w/v); (c) 0 ⁇ L/L Azadirachtin (0% v/v); 10 mg/mL U+2-ACTX-Hv1a (1% w/v); and (d) 80 ⁇ L/L Azadirachtin (0.008% v/
  • FIG.17 depicts a graph showing the mortality rate in Lesser mealworm (Alphitobius diaperinus) neonates on day three after being treated with the following: (a) 0 mg/mL U+2-ACTX-Hv1a (0% w/v); 0 mg/mL boric acid (0% w/v) (control); (b) 0 mg/mL U+2-ACTX-Hv1a (0% w/v); 2.5 mg/mL boric acid (0.25% w/v); (c) 1 mg/mL U+2-ACTX- Hv1a (0.1% w/v); 0 mg/mL boric acid (0% w/v); and (d) 1 mg/mL U+2-ACTX-Hv1a (0.1% w/v); 2.5 mg/mL boric acid (0.25% w/v).
  • FIG.18 depicts a graph showing the mortality rate in Codling Moths (Cydia pomonella) neonates on day seven after being treated with the following: (a) 0 mg/mL Beauveria bassiana toxins (0% w/v); 0 mg/mL of U+2-ACTX-Hv1a (0% w/v) (control); (b) 1.2 mg/mL Beauveria bassiana toxins (0.12% w/v); 0 mg/mL of U+2-ACTX-Hv1a (0% w/v); (c) 0 mg/mL Beauveria bassiana toxins; 2 mg/mL of U+2-ACTX-Hv1a (0.2% w/v); and (d) 1.2 mg/mL Beauveria bassiana toxins (0.12% w/v); 2 mg/mL of U+2-ACTX-Hv1a
  • FIG.19 depicts a graph showing the mortality rate in Codling Moths (Cydia pomonella) neonates on day two after being treated with (a) 0 ⁇ L/L of CpGV (0% w/v); 0 mg/mL of U+2-ACTX-Hv1a (0% w/v) (control); (b) 58.5 ⁇ L/L of CpGV (0.00585% w/v); 0 mg/mL of U+2-ACTX-Hv1a (0% w/v); (c) 0 ⁇ L/L of CpGV (0% w/v); 2 mg/mL of U+2- ACTX-Hv1a (0.2% w/v); and (d) 58.5 ⁇ L/L of CpGV (0.00585% w/v); 2 mg/mL of U+2- ACTX-Hv1a (0.2% w/v); and (d) 58.5 ⁇ L/L of CpGV (0.00585% w/v); 2 mg/
  • FIG.20 depicts a graphs showing the results of a diet incorporation assay of Novaluron with U+2-ACTX-Hv1a, and evaluating mortality in corn earworm (Helicoverpa zea) after 3-days.
  • U+2 refers to U+2-ACTX-Hv1a.
  • Novaluron Concentrations of Novaluron were as follows: (a) 80 ⁇ L/L of Novaluron (0.008% w/v); (b) 8 ⁇ L/L of Novaluron (0.0008% w/v); (c) 0.8 ⁇ L/L of Novaluron (0.00008% w/v); and (d) 0 ⁇ L/L of Novaluron (0% w/v).10 ppt of Spear corresponds to 1 mg/mL (1% w/v) of U+2-ACTX-Hv1a.
  • FIG.21 depicts a graphs showing the results of a diet incorporation assay of nanoparticles and U+2-ACTX-Hv1a, and evaluating mortality in corn earworm (Helicoverpa zea) after 3-days.
  • a diet incorporation assay of nanoparticles and U+2-ACTX-Hv1a evaluating mortality in corn earworm (Helicoverpa zea) after 3-days.
  • Spear refers to U+2-ACTX-Hv1a.
  • Concentrations of nanoparticles were as follows: (a) 50 nm silica aminated (2700 ppm); (b) 50 silica (2575 ppm); (c) 20 nm silica (1177 ppm); and (d) 10 nm silica (12500 ppm).
  • U+2 corresponds to 5 ppt of, U+2-ACTX-Hv1a, i.e., 0.5 mg/mL (0.5% w/v of the total volume of the composition) of U+2-ACTX-Hv1a.
  • Proportional mortality the number of dead insects divided by the total number of insects.
  • UTC means untreated control (water).
  • FIG.22 depicts a graph showing the mortality dose response of a diet incorporation assay of cryolite and U+2-ACTX-Hv1a against corn earworm (Helicoverpa zea) after 3 days.
  • U+2 refers to U+2-ACTX-Hv1a.
  • Concentrations of nanoparticles were as follows: (a) 10000 ppm; (b) 2000 ppm; (c) 400 ppm; and (d) 0 ppm.
  • “5’- and 3’-homology arms” or “5’ and 3’ arms” or “left and right arms” refers to the polynucleotide sequences in a vector and/or targeting vector that homologously recombine with the target genome sequence and/or endogenous gene of interest in the host organism in order to achieve successful genetic modification of the host organism’s chromosomal locus.
  • “ ⁇ -CNTX-Pn1a” or “ ⁇ -CNTX-Pn1a” or “gamma-CNTX-Pn1a” or “gamma” refers to an insecticidal neurotoxin derived from the Brazilian armed spider, Phoneutria nigriventer.
  • ⁇ -CNTX-Pn1a targets the N-methyl-D-aspartate (NMDA)-subtype of ionotropic glutamate receptor (GRIN), and sodium channels.
  • NMDA N-methyl-D-aspartate
  • GRIN ionotropic glutamate receptor
  • ⁇ / ⁇ -HXTX-Hv1a or “omega/kappa-HXTX-Hv1a,” refers to the insecticidal toxin derived from the Australian Blue Mountain Funnel-web Spider, Hadronyche versuta.
  • ⁇ / ⁇ -HXTX-Hv1a is a type of ACTX peptide, i.e., a family of insecticidal ICK peptides that have been isolated from spiders belonging to the Atracinae family.
  • ⁇ / ⁇ -HXTX-Hv1a is a positive allosteric modulators of the nicotinic acetylcholine receptor, and may also be a dual antagonist to insect voltage-gated Ca 2+ channels and voltage-gated K + channels.
  • Insecticidal spider toxins are high affinity positive allosteric modulators of the nicotinic acetylcholine receptor.
  • ACTX or “ACTX peptide” or “atracotoxin” refers to a family of insecticidal ICK peptides that have been isolated from spiders belonging to the Atracinae family. One such spider is known as the Australian Blue Mountains Funnel-web Spider, which has the scientific name Hadronyche versuta. Two examples of ACTX peptides from this species are the Omega and U peptides.
  • ADN1 promoter refers to the DNA segment comprised of the promoter sequence derived from the Schizosaccharomyces pombe adhesion defective protein 1 gene.
  • Alpha-MF signal or “ ⁇ MF secretion signal” refers to a protein that directs nascent recombinant polypeptides to the secretory pathway.
  • Agriculturally-acceptable carrier covers all adjuvants, inert components, dispersants, surfactants, tackifiers, binders, etc. that are ordinarily used in pesticide formulation technology; these are well known to those skilled in pesticide formulation.
  • “Agriculturally acceptable salt” is used herein synonymously with the term “pharmaceutically acceptable salt.”
  • “Agroinfection” means a plant transformation method where DNA is introduced into a plant cell by using Agrobacteria tumefaciens or Agrobacteria rhizogenes.
  • “Alignment” refers to a method of comparing two or more sequences (e.g., nucleotide, polynucleotide, amino acid, peptide, polypeptide, or protein sequences) for the purpose of determining their relationship to each other. Alignments are typically performed by computer programs that apply various algorithms, however, it is also possible to perform an alignment by hand.
  • Alignment programs typically iterate through potential alignments of sequences and score the alignments using substitution tables, employing a variety of strategies to reach a potential optimal alignment score.
  • Commonly-used alignment algorithms include, but are not limited to, CLUSTALW (see Thompson J. D., Higgins D. G., Gibson T. J., CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice, Nucleic Acids Research 22: 4673-4680, 1994); CLUSTALV (see Larkin M.
  • an alignment will introduce “phase shifts” and/or “gaps” into one or both of the sequences being compared in order to maximize the similarity between the two sequences, and scoring refers to the process of quantitatively expressing the relatedness of the aligned sequences.
  • Alpha-MF signal or “ ⁇ MF secretion signal” refers to a protein that directs nascent recombinant polypeptides to the secretory pathway.
  • ⁇ MF secretion signal refers to a protein that directs nascent recombinant polypeptides to the secretory pathway.
  • arachnid refers to a class of arthropods. For example in some embodiments, arachnid can mean spiders, scorpions, ticks, mites, harvestmen, or solifuges.
  • Av2 or “ATX-II” or “neurotoxin 2” or “Anemonia viridis toxin 2” or ⁇ - AITX-Avd1c” refers to a toxin isolated from the venom of Anemonia sulcata.
  • Av2 polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 588.
  • Av3 refers to a polypeptide isolated from the sea anemone, Anemonia viridis, which can target receptor site 3 on ⁇ -subunit III of voltage-gated sodium channels.
  • Av3 polypeptide is an Av3 polypeptide having the amino acid sequence of SEQ ID NO: 44 (NCBI Accession No. P01535.1).
  • AVP or “Av3 variant polypeptides” refers to an Av3 polypeptide sequence and/or a polypeptide encoded by a variant Av3 polynucleotide sequence that has been altered to produce a non-naturally occurring polypeptide and/or polynucleotide sequence.
  • BAAS barley alpha-amylase signal peptide, and is an example of an ERSP.
  • One example of a BAAS is a BAAS having the amino acid sequence of SEQ ID NO:37 (NCBI Accession No. AAA32925.1).
  • Biomass refers to any measured plant product.
  • “Binary vector” or “binary expression vector” means an expression vector which can replicate itself in both E. coli strains and Agrobacterium strains. Also, the vector contains a region of DNA (often referred to as t-DNA) bracketed by left and right border sequences that is recognized by virulence genes to be copied and delivered into a plant cell by Agrobacterium.
  • t-DNA region of DNA
  • base pair refers to a molecule comprising two chemical bases bonded to one another. For example, a DNA molecule consists of two winding strands, wherein each strand has a backbone made of an alternating deoxyribose and phosphate groups.
  • Attached to each deoxyribose is one of four bases, i.e., adenine (A), cytosine (C), guanine (G), or thymine (T), wherein adenine forms a base pair with thymine, and cytosine forms a base pair with guanine.
  • Bt toxins refers to fermentation solids, spores, and toxins produced by Bacillus thuringiensis (Bt)—a Gram positive, spore-forming bacterium, such as Bacillus thuringiensis var. kurstaki (Btk), Bacillus thuringiensis var.
  • a Bt toxin can be crystal (Cry) proteins, cytolytic (Cyt) proteins, vegetative insecticidal proteins (Vips), or other toxin produced by a Bacillus thuringiensis.
  • “Bt-resistant” or “Bt-resistance” or “Bt-resistant insect” or “Bacillus thuringiensis-toxin-resistant insects” refers to a heritable change in the sensitivity of a pest population that is reflected in the repeated failure of a product (e.g., Bt) to achieve the expected level of control when used against that pest species.
  • “C-terminal” refers to the free carboxyl group (i.e., -COOH) that is positioned on the terminal end of a polypeptide.
  • “cDNA” or “copy DNA” or “complementary DNA” refers to a molecule that is complementary to a molecule of RNA.
  • cDNA may be either single- stranded or double-stranded.
  • cDNA can be a double-stranded DNA synthesized from a single stranded RNA template in a reaction catalyzed by a reverse transcriptase.
  • cDNA refers to all nucleic acids that share the arrangement of sequence elements found in native mature mRNA species, where sequence elements are exons and 3’ and 5’ non-coding regions. Normally mRNA species have contiguous exons, with the intervening introns removed by nuclear RNA splicing, to create a continuous open reading frame encoding the protein.
  • cDNA refers to a DNA that is complementary to and derived from an mRNA template.
  • CEW refers to Corn earworm.
  • “Cleavable Linker” see Linker.
  • “Cloning” refers to the process and/or methods concerning the insertion of a DNA segment (e.g., usually a gene of interest, for example tvp) from one source and recombining it with a DNA segment from another source (e.g., usually a vector, for example, a plasmid) and directing the recombined DNA, or “recombinant DNA” to replicate, usually by transforming the recombined DNA into a bacteria or yeast host.
  • a DNA segment e.g., usually a gene of interest, for example tvp
  • a DNA segment from another source e.g., usually a vector, for example, a plasmid
  • Coding sequence refers to a polynucleotide or nucleic acid sequence that can be transcribed (e.g., in the case of DNA) or translated (e.g., in the case of mRNA) into a peptide, polypeptide, or protein, when placed under the control of appropriate regulatory sequences and in the presence of the necessary transcriptional and/or translational molecular factors.
  • coding sequence The boundaries of the coding sequence are determined by a translation start codon at the 5’ (amino) terminus and a translation stop codon at the 3’ (carboxy) terminus.
  • a transcription termination sequence will usually be located 3’ to the coding sequence.
  • a coding sequence may be flanked on the 5’ and/or 3’ ends by untranslated regions.
  • a coding sequence can be used to produce a peptide, a polypeptide, or a protein product.
  • the coding sequence may or may not be fused to another coding sequence or localization signal, such as a nuclear localization signal.
  • the coding sequence may be cloned into a vector or expression construct, may be integrated into a genome, or may be present as a DNA fragment.
  • Codon optimization refers to the production of a gene in which one or more endogenous, native, and/or wild-type codons are replaced with codons that ultimately still code for the same amino acid, but that are of preference in the corresponding host.
  • “Combination” refers to any association between two or among more items. The association can be spatial, temporal, and/or refer to the use of the two or more items for a common purpose.
  • a combination can be any spatiotemporal association, mixture, or permutation of: (1) one or more CRIPs, or pharmaceutically acceptable salt thereof; CRIP-insecticidal proteins, or pharmaceutically acceptable salt thereof; or combination thereof; and (2) one or more Insecticidal Agents (IA), as described herein, wherein (1) and (2) are used for the common purpose controlling or combating insect pests, such that the insect pest either dies, stops, or slows its movement; stops or slows its feeding; stops or slows its growth; becomes confused (e.g., with regard to navigation, locating food, sleeping behaviors, and/or mating); fails to pupate; interferes with reproduction; and/or precludes the insect from producing offspring and/or precludes the insect from producing fertile offspring.
  • IA Insecticidal Agents
  • the term “combination” may include simultaneous, separate, or sequentially administration of: (1) one or more CRIPs, or pharmaceutically acceptable salts thereof; one or more CRIP-insecticidal proteins, or pharmaceutically acceptable salts thereof; or a combination thereof; with (2) one or more Insecticidal Agents (IA), in any order of sequence.
  • IA Insecticidal Agents
  • (1) one or more CRIPs, or pharmaceutically acceptable salt thereof; CRIP-insecticidal proteins, or pharmaceutically acceptable salt thereof; or combination thereof; and (2) one or more Insecticidal Agents (IA), are considered to be administered as a “combination or “in combination” whenever a pest or the locus of a pest is exposed to, or a locus to be protected from a pest (e.g., a plant) is treated with, a simultaneous exposure to both (1) and (2).
  • each of the (1) one or more CRIPs, or pharmaceutically acceptable salt thereof; CRIP-insecticidal proteins, or pharmaceutically acceptable salt thereof; or combination thereof; and (2) one or more Insecticidal Agents (IA), may be administered sequentially or according to a different schedule—indeed, it is not required that individual doses of different agents be administered at the same time, or in the same composition.
  • “combination” refers to simultaneous administration of: (1) one or more CRIPs, or pharmaceutically acceptable salts thereof; one or more CRIP- insecticidal proteins, or pharmaceutically acceptable salts thereof; or a combination thereof; with (2) one or more Insecticidal Agents (IA).
  • “combination” refers to separate administration of: (1) one or more CRIPs, or pharmaceutically acceptable salts thereof; one or more CRIP- insecticidal proteins, or pharmaceutically acceptable salts thereof; or a combination thereof; with (2) one or more Insecticidal Agents (IA).
  • “combination” refers to sequential administration of (1) one or more CRIPs, or pharmaceutically acceptable salts thereof; one or more CRIP- insecticidal proteins, or pharmaceutically acceptable salts thereof; or a combination thereof; with (2) one or more Insecticidal Agents (IA), in any order.
  • the delay in administering the second component should not be such as to lose the beneficial effect of the combination in its entirety, i.e., the combination of (1) one or more CRIPs, or pharmaceutically acceptable salts thereof; one or more CRIP-insecticidal proteins, or pharmaceutically acceptable salts thereof; or a combination thereof; with (2) one or more Insecticidal Agents (IA).
  • IA Insecticidal Agents
  • the one or more CRIPs, or pharmaceutically acceptable salts thereof; one or more CRIP-insecticidal proteins, or pharmaceutically acceptable salts thereof; or a combination thereof can be administered on the same day as the one or more Insecticidal Agents (IA).
  • the one or more CRIPs, or pharmaceutically acceptable salts thereof; one or more CRIP-insecticidal proteins, or pharmaceutically acceptable salts thereof; or a combination thereof may be administered in the same week, or the same month as the one or more Insecticidal Agents (IA).
  • a combination can be a “mixture.”
  • “mixture” refers to a combination of two or more agents, e.g., (1) one or more CRIPs, or pharmaceutically acceptable salts thereof; one or more CRIP-insecticidal proteins, or pharmaceutically acceptable salts thereof; or a combination thereof; with (2) one or more Insecticidal Agents (IA), that are in physical and/or chemical contact with one another.
  • IA Insecticidal Agents
  • “Complementary” refers to the topological compatibility or matching together of interacting surfaces of two polynucleotides as understood by those of skill in the art.
  • a first polynucleotide is complementary to a second polynucleotide if the nucleotide sequence of the first polynucleotide is substantially identical to the nucleotide sequence of the polynucleotide binding partner of the second polynucleotide, or if the first polynucleotide can hybridize to the second polynucleotide under stringent hybridization conditions.
  • the polynucleotide whose sequence 5′-TATAC-3′ is complementary to a polynucleotide whose sequence is 5′- GTATA-3′.
  • “Conditioned medium” means the cell culture medium which has been used by cells and is enriched with cell derived materials but does not contain cells.
  • “Cone shell” or “cone snails” or “cones” refers to organisms belonging to the Conus genus of predatory marine gastropods.
  • a cone shell can be one of the following species: Conus amadis; Conus catus; Conus ermineus; Conus geographus; Conus gloriamaris; Conus kinoshitai; Conus magus; Conus marmoreus; Conus purpurascens; Conus stercusmuscarum; Conus striatus; Conus textile; or Conus tulipa.
  • Conus amadis Conus catus
  • Conus ermineus Conus geographus
  • Conus gloriamaris Conus kinoshitai
  • Conus magus Conus marmoreus
  • Conus purpurascens Conus stercusmuscarum
  • Conus textile or Conus tulipa.
  • Conus tulipa refers to the toxins isolated from cone shells that act by interfering with neuronal communication.
  • a conotoxin can be an ⁇ -, ⁇ -, ⁇ -,
  • the ⁇ -conotoxins target nicotinic ligand gated channels; ⁇ -conotoxins target voltage-gated calcium channels; ⁇ -conotoxins target the voltage-gated sodium channels; ⁇ -conotoxins target the voltage-gated sodium channel; and ⁇ -conotoxins target the voltage-gated potassium channel.
  • “Copy number” refers to the number of identical copies of a vector, an expression cassette, an amplification unit, a gene or indeed any defined nucleotide sequence, that are present in a host cell at any time.
  • a gene or another defined chromosomal nucleotide sequence may be present in one, two, or more copies on the chromosome.
  • An autonomously replicating vector may be present in one, or several hundred copies per host cell.
  • CRIP refers to Cysteine Rich Insecticidal Peptide. CRIPs are peptides rich in cysteine residues that, in some embodiments, are operable to form disulfide bonds between such cysteine residues. In some embodiments, CRIPS contain at least four (4), sometimes six (6), and sometimes eight (8) cysteine amino acids among proteins or peptides having at least 10 amino acids where the cysteines form two (2), three (3) or four (4) disulfide bonds.
  • a CRIP may or may not comprise an inhibitor cystine knot (ICK) motif.
  • ICK inhibitor cystine knot
  • a CRIP with an ICK motif can be an ACTX peptide from a spider; in other embodiments, a CRIP without an ICK motif, i.e., a non-ICK CRIP, can be a peptide like Av2 and Av3, peptides isolated from sea anemones.
  • Non-ICK CRIPS can have 4-8 cysteines which form 2-4 disulfide bonds. These cysteine- cysteine disulfide bonds stabilized toxic peptides (CRIPS) can have remarkable stability when exposed to the environment. Many CRIPS are isolated from venomous animals such as spiders, scorpions, snakes and sea snails and sea anemones and they are toxic to insects. [00100] “CRIP construct” refers to the three-dimensional arrangement/orientation of peptides, polypeptides, and/or motifs of operably linked polypeptide segments (e.g., a CRIP- insecticidal protein).
  • a CRIP expression ORF can include one or more of the following components or motifs: a CRIP; an endoplasmic reticulum signal peptide (ERSP); a linker peptide (L); a translational stabilizing protein (STA); or any combination thereof.
  • CRIP construct is used to describe the designation and/or orientation of the structural motif. In other words, the CRIP construct describes the arrangement and orientation of the components or motifs contained within a given CRIP expression ORF.
  • a CRIP construct describes, without limitation, the orientation of one of the following CRIP-insecticidal proteins: ERSP-CRIP; ERSP-(CRIP)N; ERSP-CRIP-L; ERSP-(CRIP)N-L; ERSP-(CRIP-L)N; ERSP-L-CRIP; ERSP- L-(CRIP)N; ERSP-(L-CRIP)N; ERSP-STA-CRIP; ERSP-STA-(CRIP)N; ERSP-CRIP-STA; ERSP-(CRIP) N -STA; ERSP-(STA-CRIP) N ; ERSP-(CRIP-STA) N ; ERSP-(CRIP-STA) N ; ERSP-(CRIP-STA) N ; ERSP-(CRIP-STA) N ; ERSP-(CRIP-STA) N ; ERSP-(CRIP-STA) N
  • CRIP ORF diagram refers to the composition of one or more CRIP ORFs, as written out in diagram or equation form.
  • a “CRIP ORF diagram” can be written out as using acronyms or short-hand references to the DNA segments contained within the ORF.
  • a “CRIP ORF diagram” may describe the polynucleotide segments encoding the ERSP, L, STA, and CRIP, by diagramming in equation form the DNA segments as “ersp” (i.e., the polynucleotide sequence that encodes the ERSP polypeptide); “linker” or “L” (i.e., the polynucleotide sequence that encodes the LINKER polypeptide); “sta” (i.e., the polynucleotide sequence that encodes the STA polypeptide), and “crip” (i.e., the polynucleotide sequence encoding a CRIP), respectively.
  • CRIP polynucleotide refers to a polynucleotide or group of polynucleotides operable to express and/or encode an insecticidal protein comprising one or more CRIPs in addition to one or more non-CRIP polypeptides or proteins.
  • CRIP-insecticidal protein refers to any protein, peptide, polypeptide, amino acid sequence, configuration, or arrangement, consisting of: (1) at least one CRIP, or two or more CRIPs; and (2) additional peptides, polypeptides, or proteins, wherein said additional peptides, polypeptides, or proteins have the ability to do one or more of the following: (a) increase the mortality and/or inhibit the growth of insects when the insects are exposed to a CRIP-insecticidal protein, relative to a CRIP alone; (b) increase the expression of said CRIP- insecticidal protein, e.g., in a host cell or an expression system; and/or (c) affect the post- translational processing of the CRIP-insecticidal protein.
  • an insecticidal protein can comprise a one or more CRIPs as disclosed herein.
  • a CRIP-insecticidal protein can be a polymer comprising two or more CRIPs.
  • the insecticidal protein can comprise a CRIP homopolymer, e.g., two or more CRIP monomers that are the same CRIP.
  • the insecticidal protein can comprise a CRIP heteropolymer, e.g., two or more CRIP monomers, wherein the CRIP monomers are different.
  • a CRIP-insecticidal protein can be a polymer of amino acids that when properly folded or in its most natural thermodynamic state exerts an insecticidal activity against one or more insects.
  • a CRIP-insecticidal protein can be a polymer comprising two or more CRIPs, wherein the CRIPs are operably linked via a linker peptide, e.g., a cleavable and/or non-cleavable linker.
  • a CRIP-insecticidal protein can refer to a one or more CRIPs operably linked with one or more proteins such as a stabilizing domain (STA); an endoplasmic reticulum signaling protein (ERSP); an insect cleavable or insect non-cleavable linker (L); and/or any other combination thereof.
  • a CRIP- insecticidal protein can be a non-naturally occurring protein comprising (1) a wild-type CRIP protein; and (2) additional peptides, polypeptides, or proteins, e.g., an ERSP; a linker; a STA; a UBI; or a histidine tag or similar marker.
  • “Culture” or “cell culture” refers to the maintenance of cells in an artificial, in vitro environment.
  • “Culturing” refers to the propagation of organisms on or in various kinds of media.
  • the term “culturing” can mean growing a population of cells under suitable conditions in a liquid or solid medium.
  • culturing refers to fermentative recombinant production of a heterologous polypeptide of interest and/or other desired end products (typically in a vessel or reactor).
  • “Cystine” refers to an oxidized cysteine-dimer.
  • Cystines are sulfur-containing amino acids obtained via the oxidation of two cysteine molecules, and are linked with a disulfide bond.
  • “Defined medium” means a medium that is composed of known chemical components but does not contain crude proteinaceous extracts or by-products such as yeast extract or peptone.
  • “Degeneracy” or “codon degeneracy” refers to the phenomenon that one amino acid can be encoded by different nucleotide codons. Thus, the nucleic acid sequence of a nucleic acid molecule that encodes a protein or polypeptide can vary due to degeneracies.
  • Desmethyllimocin B refers to [(5R,7R,8R,9R,10R,13S,17S)-17-[(3R)-5- Hydroxyoxolan-3-yl]-4,4,8,10,13-pentamethyl-3,16-dioxo-6,7,9,11,12,17-hexahydro-5H- cyclopenta[a]phenanthren-7-yl] acetate.
  • DNA refers to deoxyribonucleic acid, comprising a polymer of one or more deoxyribonucleotides or nucleotides (i.e., adenine [A], guanine [G], thymine [T], or cytosine [C]), which can be arranged in single-stranded or double-stranded form.
  • deoxyribonucleic acid comprising a polymer of one or more deoxyribonucleotides or nucleotides (i.e., adenine [A], guanine [G], thymine [T], or cytosine [C]), which can be arranged in single-stranded or double-stranded form.
  • nucleotides creates a polynucleotide.
  • dNTPs refers to the nucleoside triphosphates that compose DNA and RNA.
  • Double expression cassette refers to two heterologous polypeptide expression cassettes contained on the same vector.
  • Double transgene peptide expression vector or “double transgene expression vector” means a yeast expression vector that contains two copies of the heterologous polypeptide expression cassette.
  • Endogenous refers to a polynucleotide, peptide, polypeptide, protein, or process that naturally occurs and/or exists in an organism, e.g., a molecule or activity that is already present in the host cell before a particular genetic manipulation.
  • Enhancer element refers to a DNA sequence operably linked to a promoter, which can exert increased transcription activity on the promoter relative to the transcription activity that results from the promoter in the absence of the enhancer element.
  • ER or “Endoplasmic reticulum” is a subcellular organelle common to all eukaryotes where some post translation modification processes occur.
  • ERSP or “endoplasmic reticulum signal peptide” is an N-terminus sequence of amino acids that—during protein translation of the mRNA molecule encoding a CRIP—is recognized and bound by a host cell signal-recognition particle, which moves the protein translation ribosome/mRNA complex to the ER in the cytoplasm. The result is the protein translation is paused until it docks with the ER where it continues and the resulting protein is injected into the ER.
  • ersp refers to a polynucleotide encoding the peptide, ERSP.
  • ER trafficking means transportation of a cell expressed protein into ER for post-translational modification, sorting and transportation.
  • Excipient refers to any pharmacologically inactive, natural, or synthetic, component or substance that is formulated alongside (e.g., concomitantly), or subsequent to, the active ingredient of the present invention (i.e., a CRIP or CRIP-insecticidal protein).
  • an excipient can be any additive, adjuvant, binder, bulking agent, carrier, coating, diluent, disintegrant, filler, glidant, lubricant, preservative, vehicle, or combination thereof, with which a CRIP or CRIP-insecticidal protein of the present invention can be administered, and or which is useful in preparing a composition of the present invention.
  • Excipients include any such materials known in the art that are nontoxic and do not interact with other components of a composition.
  • excipients can be formulated alongside a CRIP or CRIP-insecticidal protein when preparing a composition for the purpose of bulking up compositions (thus often referred to as bulking agents, fillers or diluents).
  • an excipient can be used to confer an enhancement on the active ingredient in the final dosage form, such as facilitating absorption and/or solubility.
  • an excipient can be used to provide stability, or prevent contamination (e.g., microbial contamination).
  • an excipient can be used to confer a physical property to a composition (e.g., a composition that is a dry granular, or dry flowable powder physical form).
  • Excipient refers to (1) a DNA sequence of interest, e.g., a polynucleotide operable to encode a CRIP; and one or more of the following: (2) promoters, terminators, and/or enhancer elements; (3) an appropriate mRNA stabilizing polyadenylation signal; (4) an internal ribosome entry site (IRES); (5) introns; and/or (6) post-transcriptional regulatory elements.
  • the combination (1) with at least one of (2)-(6) is called an “expression cassette.”
  • there can be a first expression cassette comprising a polynucleotide operable to encode a CRIP.
  • there are three expression cassettes operable to encode a CRIP i.e., a triple expression cassette.
  • a double expression cassette can be generated by subcloning a second expression cassette into a vector containing a first expression cassette.
  • a triple expression cassette can be generated by subcloning a third expression cassette into a vector containing a first and a second expression cassette. Methods concerning expression cassettes and cloning techniques are well-known in the art and described herein. See also CRIP expression cassette.
  • “Expression ORF” means a nucleotide encoding a protein complex and is defined as the nucleotides in the ORF.
  • “FECT” means a transient plant expression system using Foxtail mosaic virus with elimination of coating protein gene and triple gene block.
  • “Fermentation beer” refers to spent fermentation medium, i.e., fermentation medium supernatant after removal of organisms, that has been inoculated with and consumed by a transformed host cell (e.g., a yeast cell operable to express a CRIP of the present invention).
  • fermentation beer refers to the solution that is recovered following the fermentation of the transformed host cell.
  • the term “fermentation” refers broadly to the enzymatic and anaerobic or aerobic breakdown of organic substances (e.g., a carbon substrate) nutrient substances by microorganisms under controlled conditions (e.g., temperature, oxygen, pH, nutrients, and the like) to produce fermentation products (e.g., one or more peptides of the present invention).
  • Fermentation solid(s) refers to solids (including dissolved) that remain from fermentation beer during the yeast-based fermentation process, and consists essentially of salts, complex protein source, vitamins, and additional yeast byproducts having a molecular weight cutoff of from about 200 kDa to about 1 kDa .
  • GFP means a green fluorescent protein from the jellyfish, Aequorea victoria.
  • HIS or “His” refers to histidine.
  • Homologous refers to the sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position.
  • the percent of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared ⁇ 100.
  • the term “homologous” refers to the sequence similarity between two polypeptide molecules, or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomeric subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position.
  • the homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences.
  • sequence identity refers to a measure of relatedness between two or more nucleic acids, and is given as a percentage with reference to the total comparison length. The identity calculation takes into account those nucleotide residues that are identical and in the same relative positions in their respective larger sequences.
  • homologous recombination refers to the event of substitution of a segment of DNA by another one that possesses identical regions (homologous) or nearly so.
  • homologous recombination refers to a type of genetic recombination in which nucleotide sequences are exchanged between two similar or identical molecules of DNA. Briefly, homologous recombination is most widely used by cells to accurately repair harmful breaks that occur on both strands of DNA, known as double-strand breaks.
  • homologous recombination varies widely among different organisms and cell types, most forms involve the same basic steps: after a double-strand break occurs, sections of DNA around the 5′ ends of the break are cut away in a process called resection. In the strand invasion step that follows, an overhanging 3′ end of the broken DNA molecule then “invades” a similar or identical DNA molecule that is not broken. After strand invasion, the further sequence of events may follow either of two main pathways, i.e., the double- strand break repair pathway, or the synthesis-dependent strand annealing pathway. Homologous recombination is conserved across all three domains of life as well as viruses, suggesting that it is a nearly universal biological mechanism.
  • homologous recombination can occur using a site-specific integration (SSI) sequence, whereby there is a strand exchange crossover event between nucleic acid sequences substantially similar in nucleotide composition.
  • SSI site-specific integration
  • crossover events can take place between sequences contained in the targeting construct of the invention (i.e., the SSI sequence) and endogenous genomic nucleic acid sequences (e.g., the polynucleotide encoding the peptide subunit).
  • SSI site-specific integration
  • endogenous genomic nucleic acid sequences e.g., the polynucleotide encoding the peptide subunit.
  • ICK motif or “ICK motif protein” or “inhibitor cystine knot motif” or “ICK peptides” or “cystine knot motif” or “cystine knot peptides” refers to a 16 to 60 amino acid peptide with at least 6 half-cystine core amino acids having three disulfide bridges, wherein the 3 disulfide bridges are covalent bonds and of the six half-cystine residues the covalent disulfide bonds are between the first and fourth, the second and fifth, and the third and sixth half-cystines, of the six core half-cystine amino acids starting from the N-terminal amino acid.
  • this type of peptide comprises a beta-hairpin secondary structure, normally composed of residues situated between the fourth and sixth core half-cystines of the motif, the hairpin being stabilized by the structural crosslinking provided by the motif’s three disulfide bonds.
  • additional cysteine/cystine or half-cystine amino acids may be present within the inhibitor cystine knot motif.
  • ick means a nucleotide encoding an ICK motif protein.
  • “ICK motif protein expression ORF” or “expression ORF” means a nucleotide encoding an ICK motif protein complex and is defined as the nucleotides in the ORF.
  • ICK motif protein expression vector or “ICK expression vector” or “ICK motif expression vector” means a binary vector which contains an expression ORF.
  • the binary vector also contains the necessary transcription promoter and terminator sequence surrounding the expression ORF to promote expression of the ORF and the protein it encodes.
  • Identity refers to a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing said sequences.
  • identity also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences.
  • methods to determine identity and similarity are codified in publicly available computer programs.
  • methods to determine identity and similarity between two sequences include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN, and FASTA (Altschul, S. F. et al., J. Molec.
  • BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S., et al., J. Mol. Biol.215: 403-410 (1990), the disclosures of which are incorporated herein by reference in their entireties.
  • IGER means a name for a short peptide, based on its actual sequence of one letter codes. It is an example of an intervening linker.
  • in vivo refers to the natural environment (e.g., an animal or a cell) and to processes or reactions that occur within a natural environment.
  • “Inactive” refers to a condition wherein something is not in a state of use, e.g., lying dormant and/or not working. For example, when used in the context of a gene or when referring to a gene, the term inactive means said gene is no longer actively synthesizing a gene product, having said gene product translated into a protein, or otherwise having the gene perform its normal function.
  • the term inactive can refer the failure of a gene to transcribe RNA, a failure of RNA processing (e.g., pre-mRNA processing; RNA splicing; or other post-transcriptional modifications); interference with non- coding RNA maturation; interference with RNA export (e.g., from the nucleus to the cytoplasm); interference with translation; protein folding; translocation; protein transport; and/or inhibition and/or interference with any of the molecules polynucleotides, peptides, polypeptides, proteins, transcription factors, regulators, inhibitors, or other factors that take part in any of the aforementioned processes.
  • RNA processing e.g., pre-mRNA processing; RNA splicing; or other post-transcriptional modifications
  • interference with non- coding RNA maturation e.g., from the nucleus to the cytoplasm
  • interference with RNA export e.g., from the nucleus to the cytoplasm
  • interference with translation e.g., from the
  • “Inoperable” refers to the condition of a thing not functioning, malfunctioning, or no longer able to function.
  • inoperable means said gene is no longer able to operate as it normally would, either permanently or transiently.
  • inoperable in some embodiments, means that a gene is no longer able to synthesize a gene product, having said gene product translated into a protein, or is otherwise unable to gene perform its normal function.
  • the term inoperable can refer the failure of a gene to transcribe RNA, a failure of RNA processing (e.g., pre-mRNA processing; RNA splicing; or other post-transcriptional modifications); interference with non-coding RNA maturation; interference with RNA export (e.g., from the nucleus to the cytoplasm); interference with translation; protein folding; translocation; protein transport; and/or inhibition and/or interference with any of the molecules polynucleotides, peptides, polypeptides, proteins, transcription factors, regulators, inhibitors, or other factors that take part in any of the aforementioned processes.
  • RNA processing e.g., pre-mRNA processing; RNA splicing; or other post-transcriptional modifications
  • interference with non-coding RNA maturation e.g., from the nucleus to the cytoplasm
  • interference with RNA export e.g., from the nucleus to the cytoplasm
  • interference with translation e.g., from the nucle
  • insects includes all organisms in the class “Insecta.”
  • pre-adult insects refers to any form of an organism prior to the adult stage, including, for example, eggs, larvae, and nymphs.
  • insect refers to any arthropod and nematode, including acarids, and insects known to infest all crops, vegetables, and trees and includes insects that are considered pests in the fields of forestry, horticulture and agriculture. Examples of specific crops that might be protected with the methods disclosed herein are soybean, corn, cotton, alfalfa and the vegetable crops. A list of specific crops and insects is enclosed herein.
  • Insect gut environment or “gut environment” means the specific pH and proteinase conditions found within the fore, mid or hind gut of an insect or insect larva.
  • Insect hemolymph environment means the specific pH and proteinase conditions of found within an insect or insect larva.
  • “Insecticidal activity” means that upon or after exposing the insect to compounds, agents, or peptides, the insect either dies stops or slows its movement; stops or slows its feeding; stops or slows its growth; becomes confused (e.g., with regard to navigation, locating food, sleeping behaviors, and/or mating); fails to pupate; interferes with reproduction; and/or precludes the insect from producing offspring and/or precludes the insect from producing fertile offspring.
  • Insecticidal Agent or “IA” or “Agent” refers to one or more chemical substances, molecules, nucleotides, polynucleotides, RNA, DNA, peptides, polypeptides, proteins, lipids, glycolipids, enzymes, toxins, toxicants, poisons, insecticides, pesticides, organic compounds, inorganic compounds, viruses, prokaryote organisms, or eukaryote organisms (and the agents produced from said prokaryote or eukaryote organisms).
  • an IA includes, but is not limited to, members selected from the categories of RNAi; Stomach poisons; Inhibitors of chitin biosynthesis type 0; Inhibitors of chitin biosynthesis, type 1; Insect viruses; Compounds isolated from Azadirachta indica; Compounds with unknown MOAs; Bacteria (and products therefrom); Fungi (and products therefrom); Nematodes (and products therefrom); Botanical essences; Mechanical disruptors; Fluorescent brighteners; Silica nanospheres; Chitinases; Lectins; Membrane Attack Complex/Perforin (MACPF) proteins; Plant virus coat protein-toxin fusions; Glycan binding domain/toxin fusion proteins; Acetylcholinesterase (AchE) inhibitors; GABA-gated chloride channel blockers; Sodium channel modulators; Nicotinic acetylcholine receptor (nAchR) Competitive Modulators; Nicotinic acety
  • an Insecticidal Agent can be a polymer of amino acids, a peptide, a polypeptide, or a protein; such peptide- IAs can be made and/or used in accordance with any of the methods pertaining to peptides and/or proteins as described herein.
  • “Integrative expression vector” or “integrative vector” means a yeast expression vector which can insert itself into a specific locus of the yeast cell genome and stably becomes a part of the yeast genome.
  • Intervening linker refers to a short peptide sequence in the protein separating different parts of the protein, or a short DNA sequence that is placed in the reading frame in the ORF to separate the upstream and downstream DNA sequences.
  • an intervening linker may be used allowing proteins to achieve their independent secondary and tertiary structure formation during translation.
  • the intervening linker can be either resistant or susceptible to cleavage in plant cellular environments, in the insect and/or lepidopteran gut environment, and in the insect hemolymph and lepidopteran hemolymph environment.
  • isolated refers to separating a thing and/or a component from its natural environment, e.g., a toxin isolated from a given genus or species means that toxin is separated from its natural environment, e.g., taken out of a WT organism.
  • “Kappa-ACTX peptide” refers to an excitatory toxin that inhibits insect calcium-activated potassium (KCa) channels (Slo-type).
  • Kappa-ACTX peptide can refer to peptides isolated from the Australian Blue Mountains Funnel-web Spider, Hadronyche versuta, or variants thereof.
  • kb refers to kilobase, i.e., 1000 bases.
  • the term “kb” means a length of nucleic acid molecules.
  • 1 kb refers to a nucleic acid molecule that is 1000 nucleotides long.
  • a length of double-stranded DNA that is 1 kb long contains two thousand nucleotides (i.e., one thousand on each strand).
  • a length of single- stranded RNA that is 1 kb long contains one thousand nucleotides.
  • “kDa” refers to kilodalton, a unit equaling 1,000 daltons; a “dalton” or “Da” is a unit of molecular weight (MW).
  • “Knock in” or “knock-in” or “knocks-in” or “knocking-in” refers to the replacement of an endogenous gene with an exogenous or heterologous gene, or part thereof,.
  • the term “knock-in” refers to the introduction of a nucleic acid sequence encoding a desired protein to a target gene locus by homologous recombination, thereby causing the expression of the desired protein.
  • a “knock-in” mutation can modify a gene sequence to create a loss-of-function or gain-of- function mutation.
  • knock-in can refer to the procedure by which a exogenous or heterologous polynucleotide sequence or fragment thereof is introduced into the genome (e.g., “they performed a knock-in” or “they knocked-in the heterologous gene”), or the resulting cell and/or organism (e.g., “ the cell is a “knock-in” or “the animal is a “knock-in”).
  • knock out or “knockout” or “knock-out” or “knocks-out” or “knocking-out” refers to a partial or complete suppression of the expression gene product (e.g., mRNA) of a protein encoded by an endogenous DNA sequence in a cell.
  • the “knock-out” can be effectuated by targeted deletion of a whole gene, or part of a gene encoding a peptide, polypeptide, or protein.
  • the deletion may render a gene inactive, partially inactive, inoperable, partly inoperable, or otherwise reduce the expression of the gene or its products in any cell in the whole organism and/or cell in which it is normally expressed.
  • knock-out can refer to the procedure by which an endogenous gene is made completely or partially inactive or inoperable (e.g., “they performed a knock-out” or “they knocked-out the endogenous gene”), or the resulting cell and/or organism (e.g., “ the cell is a “knock-out” or “the animal is a “knock-out”).
  • Knockdown dose 50 or “KD 50 ” refers to the median dose required to cause paralysis or cessation of movement in 50% of a population, for example a population of Musca domestica (common housefly) and/or Aedes aegypti (mosquito).
  • “l” or “linker” refers to a nucleotide encoding linker peptide.
  • “L” in the proper context refers to a linker peptide, which links a translational stabilizing protein (STA) with an additional polypeptide, e.g., a heterologous peptide, and/or multiple heterologous peptides. When referring to amino acids, “L” can also mean leucine.
  • STA translational stabilizing protein
  • “LAC4 promoter” or “Lac4 promoter” refers to a DNA segment comprised of the promoter sequence derived from the K. lactis ⁇ -galactosidase gene.
  • LAC4 promoters is strong and inducible reporter that is used to drive expression of exogenous genes transformed into yeast.
  • LAC4 terminator or “Lac4 terminator” refers to a DNA segment comprised of the transcriptional terminator sequence derived from the K. lactis ⁇ -galactosidase gene.
  • LD 20 refers to a dose required to kill 20% of a population.
  • LD50 refers to lethal dose 50 which means the dose required to kill 50% of a population.
  • Lepidopteran gut environment means the specific pH and proteinase conditions found within the fore, mid or hind gut of a lepidopteran insect or larva.
  • Linker refers to a short peptide sequence operable to link two peptides together. Linker can also refer to a short DNA sequence that is placed in the reading frame of an ORF to separate an upstream and downstream DNA sequences. In some embodiments, a linker can be cleavable by an insect protease. In some embodiments, a linker may allow proteins to achieve their independent secondary and tertiary structure formation during translation.
  • the linker can be either resistant or susceptible to cleavage in plant cellular environments, in the insect and/or lepidopteran gut environment, and/or in the insect hemolymph and lepidopteran hemolymph environment.
  • a linker can be cleaved by a protease, e.g., in some embodiments, a linker can be cleaved by a plant protease (e.g., papain, bromelain, ficin, actinidin, zingibain, and/or cardosins), an insect protease, a fungal protease, a vertebrate protease, an invertebrate protease, a bacteria protease, a mammal protease, a reptile protease, or an avian protease.
  • a plant protease e.g., papain, bromelain, ficin, actinidin, zingibain, and
  • a linker can be cleavable or non-cleavable.
  • a linker comprises a binary or tertiary region, wherein each region is cleavable by at least two types of proteases: one of which is an insect and/or nematode protease and the other one of which is a human protease.
  • a linker can have one of (at least) three roles: to cleave in the insect gut environment, to cleave in the plant cell, or to be designed not to intentionally cleave.
  • “Medium” (“plural “media”) refers to a nutritive solution for culturing cells in cell culture.
  • MW refers to the mass or weight of a molecule, and is typically measured in “daltons (Da)” or kilodaltons (kDa).
  • MW can be calculated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), analytical ultracentrifugation, or light scattering.
  • SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis
  • the SDS-PAGE method is as follows: the sample of interest is separated on a gel with a set of molecular weight standards. The sample is run, and the gel is then processed with a desired stain, followed by destaining for about 2 to 14 hours.
  • the next step is to determine the relative migration distance (Rf) of the standards and protein of interest.
  • the migration distance can be determined using the following equation: [00171]
  • the logarithm of the MW can be determined based on the values obtained for the bands in the standard; e.g., in some embodiments, the logarithm of the molecular weight of an SDS-denatured polypeptide and its relative migration distance (Rf) is plotted into a graph. After plotting the graph, interpolating the value derived will provide the molecular weight of the unknown protein band.
  • Motif refers to a polynucleotide or polypeptide sequence that is implicated in having some biological significance and/or exerts some effect or is involved in some biological process.
  • MCS Multiple cloning site
  • MCS multiple cloning site
  • “Mutant” refers to an organism, DNA sequence, amino acid sequence, peptide, polypeptide, or protein, that has an alteration or variation (for example, in the nucleotide sequence or the amino acid sequence), which causes said organism and/or sequence to be different from the naturally occurring or wild-type organism, wild-type sequence, and/or reference sequence with which the mutant is being compared.
  • this alteration or variation can be one or more nucleotide and/or amino acid substitutions or modifications (e.g., deletion or addition).
  • the one or more amino acid substitutions or modifications can be conservative; here, such a conservative amino acid substitution and/or modification in a “mutant” does not substantially diminish the activity of the mutant in relation to its non-mutant form.
  • a “mutant” possesses one or more conservative amino acid substitutions when compared to a peptide with a disclosed and/or claimed sequence, as indicated by a SEQ ID NO.
  • N-terminal refers to the free amine group (i.e., -NH 2 ) that is positioned on beginning or start of a polypeptide.
  • NCBI refers to the National Center for Biotechnology Information.
  • “nm” refers to nanometers.
  • Non-ICK CRIPS refers to peptides having 4-8 cysteines which form 2-4 disulfide bonds.
  • Non-ICK peptides include cystine knot peptides that are not ICK peptides.
  • Non-ICK peptides may have different disulfide bond connectivity patterns than ICKs.
  • Examples of a Non-ICK CRIP are peptides like Av2 and Av3, isolated from sea anemones; these anemone peptides are examples of a class of compounds that modulate sodium channels in the insect peripheral nervous system (PNS).
  • PNS insect peripheral nervous system
  • Non-Polar amino acid is an amino acid that is weakly hydrophobic and includes glycine, alanine, proline, valine, leucine, isoleucine, phenylalanine and methionine. Glycine or gly is the most preferred non-polar amino acid for the dipeptides of this invention.
  • Normalized peptide yield means the peptide yield in the conditioned medium divided by the corresponding cell density at the point the peptide yield is measured.
  • the peptide yield can be represented by the mass of the produced peptide in a unit of volume, for example, mg per liter or mg/L, or by the UV absorbance peak area of the produced peptide in the HPLC chromatograph, for example, mAu.sec.
  • the cell density can be represented by visible light absorbance of the culture at wavelength of 600 nm (OD600).
  • OD600 refers to optical density. Typically, OD is measured using a spectrophotometer. When measuring growth over time of a cell population, OD600 is preferable to UV spectroscopy; this is because at a 600 nm wavelength, the cells will not be harmed as they would under too much UV light.
  • OD660nm refers to optical densities at 660 nanometers (nm).
  • “Omega peptide” or “omega toxin,” or “omega-ACTX-Hv1a,” or “native omegaACTX-Hv1a” all refer to an ACTX peptide which was first isolated from a spider known as the Australian Blue Mountains Funnel-web Spider, Hadronyche versuta. Omega peptide is a positive allosteric modulators of the nicotinic acetylcholine receptor, and may also be a dual antagonist to insect voltage-gated Ca 2+ channels and voltage-gated K + channels.
  • Insecticidal spider toxins are high affinity positive allosteric modulators of the nicotinic acetylcholine receptor.
  • “Operable” refers to the ability to be used, the ability to do something, and/or the ability to accomplish some function or result.
  • “operable” refers to the ability of a polynucleotide, DNA sequence, RNA sequence, or other nucleotide sequence or gene to encode a peptide, polypeptide, and/or protein.
  • a polynucleotide may be operable to encode a protein, which means that the polynucleotide contains information that imbues it with the ability to create a protein (e.g., by transcribing mRNA, which is in turn translated to protein).
  • operably linked refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner.
  • operably linked can refer to two or more DNA, peptide, or polypeptide sequences.
  • operably linked can mean that the two adjacent DNA sequences are placed together such that the transcriptional activation of one DNA sequence can act on the other DNA sequence.
  • operably linked can refer to two or more peptides and/or polypeptides, wherein said two or more peptides and/or polypeptides are connected in such a way as to yield a single polypeptide chain; alternatively, the term operably linked can refer to two or more peptides that are connected in such a way that one peptide exerts some effect on the other. In yet other embodiments, operably linked can refer to two adjacent DNA sequences are placed together such that the transcriptional activation of one can act on the other.
  • ORF refers to a length of RNA or DNA sequence, between a translation start signal (e.g., AUG or ATG, respectively) and any one or more of the known termination codons, which encodes one or more polypeptide sequences.
  • the ORF describes the frame of reference as seen from the point of view of a ribosome translating the RNA code, insofar that the ribosome is able to keep reading (i.e., adding amino acids to the nascent protein) because it has not encountered a stop codon.
  • open reading frame or “ORF” refers to the amino acid sequence encoded between translation initiation and termination codons of a coding sequence.
  • initiation codon and “termination codon” refer to a unit of three adjacent nucleotides (i.e., a codon) in a coding sequence that specifies initiation and chain termination, respectively, of protein synthesis (mRNA translation).
  • an ORF is a continuous stretch of codons that begins with a start codon (usually ATG for DNA, and AUG for RNA) and ends at a stop codon (usually UAA, UAG or UGA).
  • an ORF can be length of RNA or DNA sequence, between a translation start signal (e.g., AUG or ATG) and any one or more of the known termination codons, wherein said length of RNA or DNA sequence encodes one or more polypeptide sequences.
  • an ORF can be a DNA sequence encoding a protein which begins with an ATG start codon and ends with a TGA, TAA or TAG stop codon. ORF can also mean the translated protein that the DNA encodes.
  • open reading frame and “ORF,” from the term “coding sequence,” based upon the fact that the broadest definition of “open reading frame” simply contemplates a series of codons that does not contain a stop codon. Accordingly, while an ORF may contain introns, the coding sequence is distinguished by referring to those nucleotides (e.g., concatenated exons) that can be divided into codons that are actually translated into amino acids by the ribosomal translation machinery (i.e., a coding sequence does not contain introns); however, as used herein, the terms “coding sequence”; “CDS”; “open reading frame”; and “ORF,’ are used interchangeably.
  • CDS concatenated exons
  • Out-recombined refers to the removal of a gene and/or polynucleotide sequence (e.g., an endogenous gene) that is flanked by two site- specific recombination sites (e.g., the 5’- and 3’- nucleotide sequence of a target gene that is homologous to the homology arms of a target vector) during in vivo homologous recombination.
  • a gene and/or polynucleotide sequence e.g., an endogenous gene
  • site-specific recombination sites e.g., the 5’- and 3’- nucleotide sequence of a target gene that is homologous to the homology arms of a target vector
  • Parenter crystal toxin refers to any of the peptides, polypeptides, and/or proteins that are part of the parasporal body or parasporal crystal, which is a bipyramidal crystal containing one or more peptides, polypeptides, and/or proteins.
  • this toxin-containing parasporal crystal dissolves in the alkaline gut juices, followed by cleavage via midgut proteases of the protoxin, which yields an active peptide toxin, e.g., a ⁇ -endotoxin.
  • “Peptide expression cassette” or “expression cassette” means a DNA sequence which is composed of all the DNA elements necessary to complete transcription of an insecticidal protein in a biological expression system. In the described methods herein, it includes a transcription promoter, a DNA sequence to encode an ⁇ -mating factor signal sequence, a cleavage site, an insecticidal protein transgene, a stop codon and a transcription terminator.
  • “Peptide expression vector” means a host organism expression vector which contains a heterologous peptide transgene.
  • “Peptide expression yeast strain”, “peptide expression strain” or “peptide production strain” means a yeast strain which can produce a heterologous peptide.
  • Peptide-IA refers to Insecticidal Agents that are amino acids, peptides, polypeptides, and/or proteins.
  • Peptide transgene or “insecticidal peptide transgene” or “insecticidal protein transgene” refers to a DNA sequence that encodes a peptide of interest and can be translated in a biological expression system.
  • Peptide yield means the insecticidal peptide concentration in the conditioned medium which is produced from the cells of a peptide expression yeast strain.
  • Peritrophic membrane means a lining inside the insect gut that traps large food particles can aid in their movement through the gut while allowing digestion, but also protecting the gut wall.
  • “Pest” includes, but is not limited to: insects, fungi, bacteria, nematodes, mites, ticks, and the like.
  • Pestly-effective amount refers to an amount of the pesticide that is able to bring about death to at least one pest, or to noticeably reduce pest growth, feeding, or normal physiological development. This amount will vary depending on such factors as, for example, the specific target pests to be controlled, the specific environment, location, plant, crop, or agricultural site to be treated, the environmental conditions, and the method, rate, concentration, stability, and quantity of application of the pesticidally-effective polypeptide composition. The formulations may also vary with respect to climatic conditions, environmental considerations, and/or frequency of application and/or severity of pest infestation.
  • Plant shall mean whole plants, plant tissues, plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, propagules, embryos and progeny of the same. Plant cells can be differentiated or undifferentiated (e.g. callus, suspension culture cells, protoplasts, leaf cells, root cells, phloem cells, and pollen).
  • Plant transgenic protein means a protein from a heterologous species that is expressed in a plant after the DNA or RNA encoding it was delivered into one or more of the plant cells.
  • Plant cleavable linker means a cleavable linker peptide, or a nucleotide encoding a cleavable linker peptide, which contains a plant protease recognition site and can be cleaved during the protein expression process in the plant cell.
  • Plant-incorporated protectant or “PIP” means an insecticidal protein produced by transgenic plants, and the genetic material necessary for the plant to produce the protein.
  • Plasmid refers to a DNA segment that acts as a carrier for a gene of interest and, when transformed or transfected into an organism, can replicate and express the DNA sequence contained within the plasmid independently of the host organism. Plasmids are a type of vector, and can be “cloning vectors” (i.e., simple plasmids used to clone a DNA fragment and/or select a host population carrying the plasmid via some selection indicator) or “expression plasmids” (i.e., plasmids used to produce large amounts of polynucleotides and/or polypeptides).
  • cloning vectors i.e., simple plasmids used to clone a DNA fragment and/or select a host population carrying the plasmid via some selection indicator
  • expression plasmids i.e., plasmids used to produce large amounts of polynucleotides and/or polypeptides.
  • “Polar amino acid” is an amino acid that is polar and includes serine, threonine, cysteine, asparagine, glutamine, histidine, tryptophan and tyrosine; preferred polar amino acids are serine, threonine, cysteine, asparagine and glutamine; with serine being most highly preferred.
  • “Polynucleotide” refers to a polymeric-form of nucleotides (e.g., ribonucleotides, deoxyribonucleotides, or analogs thereof) of any length; e.g., a sequence of two or more ribonucleotides or deoxyribonucleotides.
  • polynucleotide includes double- and single-stranded DNA, as well as double- and single- stranded RNA; it also includes modified and unmodified forms of a polynucleotide (modifications to and of a polynucleotide, for example, can include methylation, phosphorylation, and/or capping).
  • a polynucleotide can be one of the following: a gene or gene fragment (for example, a probe, primer, EST, or SAGE tag); genomic DNA; genomic DNA fragment; exon; intron; messenger RNA (mRNA); transfer RNA; ribosomal RNA; ribozyme; cDNA; recombinant polynucleotide; branched polynucleotide; plasmid; vector; isolated DNA of any sequence; isolated RNA of any sequence; nucleic acid probe; primer or amplified copy of any of the foregoing.
  • a gene or gene fragment for example, a probe, primer, EST, or SAGE tag
  • genomic DNA for example, genomic DNA fragment; genomic DNA fragment; exon; intron; messenger RNA (mRNA); transfer RNA; ribosomal RNA; ribozyme; cDNA; recombinant polynucleotide; branched polynucleotide; plasmid; vector; isolated DNA of
  • a polynucleotide can refer to a polymeric-form of nucleotides operable to encode the open reading frame of a gene.
  • a polynucleotide can refer to cDNA.
  • polynucleotides can have any three-dimensional structure and may perform any function, known or unknown. The structure of a polynucleotide can also be referenced to by its 5’- or 3’- end or terminus, which indicates the directionality of the polynucleotide.
  • Adjacent nucleotides in a single-strand of polynucleotides are typically joined by a phosphodiester bond between their 3’ and 5’ carbons.
  • different internucleotide linkages could also be used, such as linkages that include a methylene, phosphoramidate linkages, etc. This means that the respective 5’ and 3’ carbons can be exposed at either end of the polynucleotide, which may be called the 5’ and 3’ ends or termini.
  • the 5’ and 3’ ends can also be called the phosphoryl (PO 4 ) and hydroxyl (OH) ends, respectively, because of the chemical groups attached to those ends.
  • PO 4 phosphoryl
  • OH hydroxyl
  • any embodiment that makes or uses a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form.
  • a polynucleotide can include modified nucleotides, such as methylated nucleotides and nucleotide analogs (including nucleotides with non- natural bases, nucleotides with modified natural bases such as aza- or deaza-purines, etc.). If present, modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide.
  • a polynucleotide can also be further modified after polymerization, such as by conjugation with a labeling component. Additionally, the sequence of nucleotides in a polynucleotide can be interrupted by non-nucleotide components. One or more ends of the polynucleotide can be protected or otherwise modified to prevent that end from interacting in a particular way (e.g. forming a covalent bond) with other polynucleotides.
  • a polynucleotide can be composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); and thymine (T).
  • Uracil (U) can also be present, for example, as a natural replacement for thymine when the polynucleotide is RNA. Uracil can also be used in DNA.
  • sequence refers to the alphabetical representation of a polynucleotide or any nucleic acid molecule, including natural and non-natural bases.
  • RNA molecule refers to a polynucleotide having a ribose sugar rather than deoxyribose sugar and typically uracil rather than thymine as one of the pyrimidine bases.
  • An RNA molecule of the invention is generally single-stranded, but can also be double-stranded.
  • the RNA molecule can include the single-stranded molecules transcribed from DNA in the cell nucleus, mitochondrion or chloroplast, which have a linear sequence of nucleotide bases that is complementary to the DNA strand from which it is transcribed.
  • a polynucleotide can further comprise one or more heterologous regulatory elements.
  • the regulatory element is one or more promoters; enhancers; silencers; operators; splicing signals; polyadenylation signals; termination signals; RNA export elements, internal ribosomal entry sites (IRES); poly-U sequences; or combinations thereof.
  • IRS internal ribosomal entry sites
  • PTGS post-transcriptional gene silencing
  • PTGS means a cellular process within living cells that suppress the expression of a gene.
  • “Post-transcriptional regulatory elements” are DNA segments and/or mechanisms that affect mRNA after it has been transcribed.
  • Post-transcriptional mechanisms include splicing events, capping, addition of a Poly (A) tail, and other mechanisms known to those having ordinary skill in the art.
  • “Promoter” refers to a region of DNA to which RNA polymerase binds and initiates the transcription of a gene.
  • “Protein” has the same meaning as “peptide” and/or “polypeptide” in this document.
  • “Ratio” refers to the quantitative relation between two amounts showing the number of times one value contains or is contained within the other.
  • Reading frame refers to one of the six possible reading frames, three in each direction, of the double stranded DNA molecule.
  • the reading frame that is used determines which codons are used to encode amino acids within the coding sequence of a DNA molecule.
  • a reading frame is a way of dividing the sequence of nucleotides in a polynucleotide and/or nucleic acid (e.g., DNA or RNA) into a set of consecutive, non-overlapping triplets.
  • “Recombinant DNA” or “rDNA” refers to DNA that is comprised of two or more different DNA segments.
  • “Recombinant vector” means a DNA plasmid vector into which foreign DNA has been inserted.
  • regulatory elements refers to a genetic element that controls some aspect of the expression and/or processing of nucleic acid sequences.
  • a regulatory element can be found at the transcriptional and post- transcriptional level. Regulatory elements can be cis-regulatory elements (CREs), or trans- regulatory elements (TREs).
  • CREs cis-regulatory elements
  • TREs trans- regulatory elements
  • a regulatory element can be one or more promoters; enhancers; silencers; operators; splicing signals; polyadenylation signals; termination signals; RNA export elements, internal ribosomal entry sites (IRES); poly-U sequences; and/or other elements that influence gene expression, for example, in a tissue- specific manner; temporal-dependent manner; to increase or decrease expression; and/or to cause constitutive expression.
  • Restriction enzyme or “restriction endonuclease” refers to an enzyme that cleaves DNA at a specified restriction site.
  • a restriction enzyme can cleave a plasmid at an EcoRI, SacII or BstXI restriction site allowing the plasmid to be linearized, and the DNA of interest to be ligated.
  • “Restriction site” refers to a location on DNA comprising a sequence of 4 to 8 nucleotides, and whose sequence is recognized by a particular restriction enzyme.
  • Salannin refers to a chemical compound isolated from Azadirachta indica that has insecticidal activity.
  • Salannin has a molecular formula of C 34 H 44 O 9 , and a molecular weight of 596.7 g/mol.
  • “Sea anemone” refers to a group of marine animals of the order Actiniaria. Sea anemones are named after the anemone, which is a terrestrial flowering plant, due to colorful appearance many sea anemones possess.
  • a sea anemone is one of the following species: Actinia equine; Anemonia erythraea; Anemonia sulcata; Anemonia viridis; Anthopleura elegantissima; Anthopleura fuscoviridis; Anthopleura xanthogrammica; Bunodosoma caissarum; Bunodosoma cangicum; Bunodosoma granulifera; Heteractis crispa; Parasicyonis actinostoloides; Radianthus paumotensis; or Stoichactis helianthus.
  • “Selection gene” means a gene which confers an advantage for a genetically modified organism to grow under the selective pressure.
  • “Serovar” or “serotype” refers to a group of closely related microorganisms distinguished by a characteristic set of antigens. In some embodiments, a serovar is an antigenically and serologically distinct variety of microorganism
  • “sp.” refers to species.
  • “ssp.” or “subsp.” refers to subspecies.
  • “Subcloning” or “subcloned” refers to the process of transferring DNA from one vector to another, usually advantageous vector.
  • polynucleotide encoding a mutant or a peptide can be subcloned into a pKlac1 plasmid subsequent to selection of yeast colonies transformed with pKLAC1 plasmids.
  • SSI is an acronym that is context dependent. In some contexts, it can refer to “site-specific integration,” which is used to refer to a sequence that will permit in vivo homologous recombination to occur at a specific site within a host organism’s genome.
  • the term “site-specific integration” refers to the process directing a transgene to a target site in a host-organism’s genome, allowing the integration of genes of interest into pre-selected genome locations of a host-organism.
  • SSI can refer to “surface spraying indoors,” which is a technique of applying a variable volume sprayable volume of an insecticide onto surfaces where vectors rest, such as on walls, windows, floors and ceilings.
  • STA Translational stabilizing protein or “stabilizing domain” or “stabilizing protein” (used interchangeably herein) means a peptide or protein with sufficient tertiary structure that it can accumulate in a cell without being targeted by the cellular process of protein degradation.
  • the protein can be between 5 and 50 amino acids long.
  • the translational stabilizing protein is coded by a DNA sequence for a protein that is operably linked with a sequence encoding an insecticidal protein or a CRIP in the ORF.
  • the operably- linked STA can either be upstream or downstream of the CRIP and can have any intervening sequence between the two sequences (STA and CRIP) as long as the intervening sequence does not result in a frame shift of either DNA sequence.
  • the translational stabilizing protein can also have an activity which increases delivery of the CRIP across the gut wall and into the hemolymph of the insect.
  • sta means a nucleotide encoding a translational stabilizing protein.
  • Structural motif refers to the three-dimensional arrangement of peptides and/or polypeptides, and/or the arrangement of operably linked polypeptide segments. For example, a polypeptide having an ERSP motif, an STA motif, a LINKER motif, and a CRIP polypeptide motif, has an overall “structural motif” of ERSP-STA-L-CRIP.
  • Ta1b or “U1-agatoxin-Ta1b” or “Ta1bWT” or “wild-type U1-agatoxin- Ta1b” refers to a polypeptide isolated from the Hobo spider, Eratigena agrestis.
  • U1-agatoxin-Ta1b is a polypeptide having the amino acid sequence of SEQ ID NO:1 (NCBI Accession No. O46167.1).
  • Ta1b variant polynucleotide or “U1-agatoxin-Ta1b variant polynucleotide” refers to a polynucleotide or group of polynucleotides operable to express and/or encode an insecticidal protein comprising one or more TVPs.
  • U1-agatoxin-Ta1b variant polynucleotide when used to describe the U1-agatoxin-Ta1b variant polynucleotide sequence contained in a TVP expression ORF, its inclusion in a vector, and/or when describing the polynucleotides encoding an insecticidal protein, is described as “tvp” and/or “Tvp.”
  • tvp the polynucleotides encoding an insecticidal protein
  • Toxin refers to a venom and/or a poison, especially a protein or conjugated protein produced by certain animals, higher plants, and pathogenic bacteria.
  • toxin is reserved natural products, e.g., molecules and peptides found in scorpions, spiders, snakes, poisonous mushrooms, etc.
  • toxicant is reserved for man- made products and/or artificial products e.g., man-made chemical pesticides.
  • Transfection and “transformation” both refer to the process of introducing exogenous and/or heterologous DNA or RNA (e.g., a vector containing a polynucleotide that encodes a CRIP) into a host organism (e.g., a prokaryote or a eukaryote).
  • a host organism e.g., a prokaryote or a eukaryote
  • transformation to describe processes where exogenous and/or heterologous DNA or RNA are introduced into a bacterial cell; and reserve the term “transfection” for processes that describe the introduction of exogenous and/or heterologous DNA or RNA into eukaryotic cells.
  • transformation and “transfection” are used synonymously, regardless of whether a process describes the introduction exogenous and/or heterologous DNA or RNA into a prokaryote (e.g., bacteria) or a eukaryote (e.g., yeast, plants, or animals).
  • Transgene means a heterologous DNA sequence encoding a protein which is transformed into a plant.
  • Transgenic host cell means a cell which is transformed with a gene and has been selected for its transgenic status via an additional selection gene.
  • Transgenic plant means a plant that has been derived from a single cell that was transformed with foreign DNA such that every cell in the plant contains that transgene.
  • Transient expression system means an Agrobacterium tumefaciens-based system which delivers DNA encoding a disarmed plant virus into a plant cell where it is expressed. The plant virus has been engineered to express a protein of interest at high concentrations, up to 40% of the TSP.
  • TSP total soluble protein
  • TVP or “U1-agatoxin-Ta1b Variant Polypeptides (TVPs)” or “Ta1b Variant Polypeptides (TVPs)” refers to mutants or variants of the wild-type U1-agatoxin-Ta1b polypeptide sequence and/or a polynucleotide sequence encoding a wild-type U1-agatoxin- Ta1b polypeptide, that have been altered to produce a non-naturally occurring polypeptide and/or polynucleotide sequence.
  • An exemplary wild-type U1-agatoxin-Ta1b polypeptide sequence is provided herein, having the amino acid sequence of SEQ ID NO: 1.
  • a TVP can have an amino acid sequence according to any of the amino acid sequences listed in Table 1. Accordingly, the term “TVP” refers to peptides having one or more mutations relative to the amino acid sequence set forth in SEQ ID NO: 1.
  • a TVP can have an amino acid sequence according to Formula (I): E-P-D-E-I-C-R-X 1 -X 2 -M-X 3 -N-K-E-F-T-Y-X 4 -S-N-V-C-N-N-C-G-D-Q-V-A-A-C-E-A-E- C-F-X 5 -N-D-V-Y-Z 1 -A-C-H-E-A-Q-X 6 -X 7
  • a TVP can have an amino acid sequence according to Formula (II): E-P-D-E-I-C-R-A-X 1 -M-T-N-K-E-F-T-Y-K-S-N-V-C-N-N-C-G-D-Q-V-A-A-C-E-A-E- C-F-R-N-D-V-Y-Z 1 -A-C-H-E-A-Q-K-G Formula (II) [00252] wherein the polypeptide comprises at least one amino acid substitution relative to the wild-type sequence of U1-agatoxin-Ta1b as set forth in SEQ ID NO:1, and wherein X 1 is R or Q; and Z 1 is T or A; or a pharmaceutically acceptable salt thereof.
  • U-ACTX-Hv1a or “hybrid peptide” or “hybrid toxin” or “hybrid-ACTX- Hv1a” or “native hybridACTX-Hv1a” or “U peptide” or “U toxin” or “native U” or “native U-ACTX-Hv1a,” all refer to an ACTX peptide, which was discovered from a spider known as the Australian Blue Mountains Funnel-web Spider, Hadronyche versuta.
  • U-ACTX-Hv1a is a positive allosteric modulators of the nicotinic acetylcholine receptor, and may also be a dual antagonist to insect voltage-gated Ca 2+ channels and voltage-gated K + channels. See Chambers et al., Insecticidal spider toxins are high affinity positive allosteric modulators of the nicotinic acetylcholine receptor. FEBS Lett.2019 Jun; 593(12):1336-1350; and Windley et al., Lethal effects of an insecticidal spider venom peptide involve positive allosteric modulation of insect nicotinic acetylcholine receptors.
  • U-ACTX-Hv1a peptide is provided in SEQ ID NO: 60.
  • U+2 peptide or “U+2 protein” or “U+2 toxin” or “U+2” or “U+2-ACTX- Hv1a” or “Spear” all refer to a U-ACTX-Hv1a having an additional dipeptide operably linked to the native peptide.
  • the additional dipeptide that is operably linked to the U peptide is indicated by the “+2” or “plus 2” can be selected from among several peptides, any of which may result in a “U+2 peptide” with unique properties as discussed herein.
  • the dipeptide is “GS”; an exemplary U+2-ACTX-Hv1a peptide is set forth in SEQ ID NO: 61.
  • UBI refers to ubiquitin.
  • UBI can refer to a ubiquitin monomer isolated from Zea mays.
  • var.” refers to varietas or variety.
  • variable is used to indicate a taxonomic category that ranks below the species level and/or subspecies (where present). In some embodiments, the term “var.” represents members differing from others of the same subspecies or species in minor but permanent or heritable characteristics. [00257] “Variant” or “variant sequence” or “variant peptide” refers to an amino acid sequence that possesses one or more conservative amino acid substitutions or conservative modifications. The conservative amino acid substitutions in a “variant” does not substantially diminish the activity of the variant in relation to its non-varied form.
  • a “variant” possesses one or more conservative amino acid substitutions when compared to a peptide with a disclosed and/or claimed sequence, as indicated by a SEQ ID NO.
  • Vector refers to the DNA segment that accepts a foreign gene of interest (e.g., crip). The gene of interest is known as an “insert” or “transgene.”
  • Vip or “VIP” or “Vegetative Insecticidal Proteins” refer to proteins discovered from screening the supernatant of vegetatively grown strains of Bt for possible insecticidal activity. Vips have little or no similarity to Cry proteins.
  • Vipification refers to a process of converting a material into a glass-like amorphous material.
  • the glass-like amorphous solid may be free of any crystalline structure. Solidification of a vitreous solid occurs at the glass transition temperature (Tg).
  • Wild type or “WT” refers to the phenotype and/or genotype (i.e., the appearance or sequence) of an organism, polynucleotide sequence, and/or polypeptide sequence, as it is found and/or observed in its naturally occurring state or condition.
  • Yeast expression vector or “expression vector” or “vector” means a plasmid which can introduce a heterologous gene and/or expression cassette into yeast cells to be transcribed and translated.
  • Yield refers to the production of a peptide, and increased yields can mean increased amounts of production, increased rates of production, and an increased average or median yield and increased frequency at higher yields.
  • yield when used in reference to plant crop growth and/or production, as in “yield of the plant” refers to the quality and/or quantity of biomass produced by the plant.
  • CRIPs CYSTEINE RICH INSECTICIDAL PROTEINS
  • IA Insecticidal Agents
  • CRIPs of the present invention which can be used in combination with the Insecticidal Agents (IAs) of the present invention, are described in detail below. All CRIPs suitable for the combinations of the present invention and contemplated below include CRIP-insecticidal proteins.
  • a CRIP can be a spider toxin peptide or protein isolated from one of the following: Phoneutria nigriventer; Allagelena opulenta; Cupiennius salei; Plectreurys tristis; Coremiocnemis valida; Haplopelma huwenum; Agelena orientalis; Allagelena opulenta; Segestria florentina; Apomastus schlingeri; Phoneutria keyserlingi; Macrothele gigas; Macrothele raveni; Missulena bradleyi; Pireneitega luctuosa; Phoneutria reidyi; Illawara wisharti; Eucratoscelus constrictus; Agelenopsis aperta; Hololena curta; Oxyopes lineatus; Brachypelma albicep
  • a CRIP can be isolated from Hadronyche versuta, or the Blue Mountain funnel web spider, Hadronyche venenata, Atrax robustus, Atrax formidabilis, or Atrax infensus.
  • a CRIP can be any of the following spider peptides, polypeptides, and/or toxins: U+2-ACTX-Hv1a; ⁇ -CNTX-Pn1a ; U13-ctenitoxin-Pn1a, U13- ctenitoxin-Pn1b,U13-ctenitoxin-Pn1c, U1-agatoxin-Aop1a, U1-ctenitoxin-Cs1a, U1- nemetoxin-Csp1a, U1-nemetoxin-Csp1b, U1-nemetoxin-Csp1c, U1-plectoxin-Pt1a, U1- plectoxin-Pt1b, U1-plectoxin-Pt1c, U1-plectoxin-Pt1d, U1-plectoxin-Pt1f, U1-theraphotoxin- Cv1
  • a CRIP can be a spider toxin peptide or protein having an amino acid sequence as set forth in any one of SEQ ID NOs: 192-278 and 281-370.
  • a polynucleotide encoding a CRIP can encode a CRIP having an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6%
  • ACTX Peptides [00277]
  • a CRIP can be an ACTX peptide.
  • a CRIP can be one or more of the following ACTX peptides: U-ACTX-Hv1a, U+2-ACTX-Hv1a, rU-ACTX-Hv1a, rU-ACTX-Hv1b, r ⁇ -ACTX- Hv1c, ⁇ -ACTX-Hv1a, and/or ⁇ -ACTX-Hv1a+2.
  • Exemplary ACTX peptides include: U-ACTX-Hv1a, having the amino acid sequence (SEQ ID NO: 60); U+2-ACTX-Hv1a, having the amino acid sequence (SEQ ID NO: 61); Omega-ACTX-Hv1a, having the amino acid sequence (SEQ ID NO: 62); “ ⁇ +2- ACTX-Hv1a+2” (or Omega+2-ACTX-Hv1a) having the amino acid sequence (SEQ ID NO: 63); and Kappa+2-ACTX-Hv1a (or ⁇ +2-ACTX-Hv1a), having the amino acid sequence (SEQ ID NO: 64).
  • a CRIP can be “Kappa-ACTX-Hv1a” (or ⁇ +2-ACTX- Hv1a) having the amino acid sequence (SEQ ID NO: 594).
  • an ACTX peptide may comprise an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% amino acid sequence identity to SEQ ID NOs: 60-64, 192
  • a polynucleotide encoding an ACTX peptide can encode an ACTX peptide having an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence set forth in SEQ ID NOs: 60-64, and 5
  • ⁇ -CNTX-Pn1a peptides [00284]
  • a CRIP can be a ⁇ -CNTX-Pn1a or ⁇ -CNTX- Pn1a toxin.
  • the ⁇ -CNTX-Pn1a peptide is an insecticidal neurotoxin derived from the Brazilian armed spider, Phoneutria nigriventer.
  • ⁇ -CNTX-Pn1a targets the N-methyl-D- aspartate (NMDA)-subtype of ionotropic glutamate receptor (GRIN), and sodium channels.
  • NMDA N-methyl-D- aspartate
  • GRIN ionotropic glutamate receptor
  • An exemplary wild-type full length ⁇ -CNTX-Pn1a peptide has an amino acid sequence of: (SEQ ID NO: 689) (NCBI Accession No. P59367).
  • a recombinant mature ⁇ -CNTX-Pn1a peptide is provided, having an amino acid sequence of (SEQ ID NO: 65).
  • an ⁇ -CNTX-Pn1a peptide may comprise an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% amino acid sequence identity to SEQ ID NO: 65.
  • a polynucleotide encoding a ⁇ -CNTX-Pn1a peptide can encode a ⁇ -CNTX-Pn1a peptide having an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence that is at least 50% identical
  • Hobo spiders (Eratigena agrestis, formerly Tegenaria agrestis) are venomous spiders that are members of the Agelenidae family of spiders, or funnel web weavers. See Ingale A, Antigenic epitopes prediction and MHC binder of a paralytic insecticidal toxin (ITX-1) of Tegenaria agrestis (hobo spider).4 August 2010 Volume 2010:2 pp 97-103. The venom of Hobo spiders has been implicated as possessing insecticidal activity.
  • Agatoxins are a chemically diverse group of toxins that can induce various insecticidal effects depending on the target species; .e.g., agatoxins cause slow-onset spastic paralysis in coleopterans, lepidopterans, and dipterans; increase the rate of neuron firing in the central nervous system (CNS) of houseflies (Musca domestica); and are lethal to other insects (e.g., the blowfly, Lucilia cuprina). Accordingly, agatoxins are implicated in targeting the CNS. See Undheim et al., Weaponization of a hormone: convergent recruitment of hyperglycemic hormone into the venom of arthropod predators.
  • agatoxins include U1-agatoxin-Ta1a and U1-agatoxin-Ta1b, which are both members of the helical arthropod-neuropeptide-derived (HAND) toxins family. In addition to spiders, these toxins can also be found in the venom of centipedes.
  • the agatoxins are evolutionary offshoots of an ancient ecdysozoan hormone family, i.e., the ion transport peptide/crustacean hyperglycemic hormone (ITP/CHH) family. See Undheim et al., Weaponization of a hormone: convergent recruitment of hyperglycemic hormone into the venom of arthropod predators. Structure 23: 1283-1292, and Johnson et al., Novel insecticidal peptides from Tegenaria agrestis spider venom may have a direct effect on the insect central nervous system. Arch. Insect Biochem. Physiol.38:19-31(1998).
  • the Hobo-spider-derived U1-agatoxin-Ta1b toxin has a full amino acid sequence of positions 1-17, and the mature toxin from positions 18-68. Id.
  • the protein comprises four tightly packed ⁇ -helices, with no ⁇ -strands present, and the molecular mass of the mature toxin is 5700.39 Daltons (Da). Id.
  • An exemplary mature wild-type U1-agatoxin-Ta1b polypeptide from Eratigena agrestis is provided having the amino acid sequence: (SEQ ID NO:1).
  • the mature wild-type U1-agatoxin-Ta1b toxin undergoes an excision event of the C-terminal glycine, yielding the following amino acid sequence: (SEQ ID NO: 60).
  • a subsequent post-translational event result in the mature wild-type U1-agatoxin- Ta1b toxin having a C-terminal amidation.
  • U1-agatoxin-Ta1b Variant Polypeptides are mutants or variants that differ from the wild-type U1-agatoxin-Ta1b (SEQ ID NO:1) in some way, e.g., in some embodiments, this variance can be an amino acid substitution, deletion, or addition; or a change to the polynucleotide encoding the wild-type U1-agatoxin-Ta1b resulting in an amino acid substitution, deletion, or addition.
  • a TVP can have an amino acid sequence according to SEQ ID NOs: 2-15, 49-53, 621-622, 624-628, 631-640, 642-651, or 653-654, as shown in Table 1. [00296] Table 1. TVPs of the present invention.
  • a polynucleotide sequence having a sequence according to SEQ ID NOs: 2-15, 49-53, 621-622, 624-628, 631-640, 642-651, or 653-654 is operable to encode a TVP.
  • a polynucleotide as shown in Table 2 is operable to encode a TVP.
  • Table 2 is operable to encode a TVP.
  • a TVP comprises one or more mutations relative to the wild-type sequence of U1-agatoxin-Ta1b as set forth in SEQ ID NO:1.
  • a TVP can have a first, second, or third mutation relative to the wild-type sequence of U1-agatoxin-Ta1b as set forth in SEQ ID NO:1.
  • an insecticidal U 1 -agatoxin-Ta1b variant polypeptide can be a TVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence to the amino acid sequence according to Formula (I
  • an insecticidal U 1 -agatoxin-Ta1b variant polypeptide can be a TVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence to the amino acid sequence according to Formula (I)
  • an insecticidal U 1 -agatoxin-Ta1b variant polypeptide can be a TVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence to the amino acid sequence according to Formula (I)
  • an insecticidal U 1 -agatoxin-Ta1b variant polypeptide can be a TVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence to the amino acid sequence according to Formula (I)
  • an insecticidal U 1 -agatoxin-Ta1b variant polypeptide can be a TVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence to the amino acid sequence according to Formula (I
  • an insecticidal U 1 -agatoxin-Ta1b variant polypeptide can be a TVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence to the amino acid sequence according to Formula (I
  • an insecticidal U 1 -agatoxin-Ta1b variant polypeptide can be a TVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence to the amino acid sequence according to Formula (I
  • an insecticidal U 1 -agatoxin-Ta1b variant polypeptide can be a TVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence to the amino acid sequence according to Formula (I
  • an insecticidal U 1 -agatoxin-Ta1b variant polypeptide can be a TVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence to the amino acid sequence according to Formula (I)
  • an insecticidal U 1 -agatoxin-Ta1b variant polypeptide can be a TVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence to the amino acid sequence according to Formula (I
  • an insecticidal U 1 -agatoxin-Ta1b variant polypeptide can be a TVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99
  • an insecticidal U 1 -agatoxin-Ta1b variant polypeptide can be a TVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence to the amino acid sequence “EPDEICRA
  • an insecticidal U 1 -agatoxin-Ta1b variant polypeptide can be a TVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence to the amino acid sequence according to Formula (I
  • an insecticidal U 1 -agatoxin-Ta1b variant polypeptide can be a TVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence to the amino acid sequence according to Formula (II
  • an insecticidal U 1 -agatoxin-Ta1b variant polypeptide can be a TVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence to the amino acid sequence according to Formula (I
  • an insecticidal U 1 -agatoxin-Ta1b variant polypeptide can be a TVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence to the amino acid sequence as set forth in any
  • the TVP may comprise an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence: (SEQ ID NO: 51).
  • a TVP can be a (SEQ ID NO: 51).
  • polynucleotides encoding TVPs can be used to transform plant cells, yeast cells, or bacteria cells.
  • the insecticidal TVP transgenic proteins may be formulated into compositions that can be sprayed or otherwise applied in any manner known to those skilled in the art to the surface of plants or parts thereof. Accordingly, DNA constructs are provided herein, operable to encode one or more TVPs under the appropriate conditions in a host cell, for example, a plant cell.
  • Methods for controlling a pest infection by a parasitic insect of a plant cell comprises administering or introducing a polynucleotide encoding a TVP as described herein to a plant, plant tissue, or a plant cell by recombinant techniques and growing said recombinantly altered plant, plant tissue or plant cell in a field exposed to the pest.
  • TVPs can be formulated into a sprayable composition consisting of a TVP and an excipient, and applied directly to susceptible plants by direct application, such that upon ingestion of the TVP by the infectious insect results in a deleterious effect.
  • a CRIP can be any of the following scorpion peptides, polypeptides, and/or toxins: Imperatoxin-A (IpTxa), Potassium channel toxin alpha-KTx 10.2 (Cobatoxin-2), Potassium channel toxin alpha-KTx 11.1 (Parabutoxin-1), Potassium channel toxin alpha-KTx 11.2 (Parabutoxin-2), Potassium channel toxin alpha-KTx 11.3 (Parabutoxin-10), Potassium channel toxin alpha-KTx 12.1 (Butantoxin), Potassium channel toxin alpha-KTx 12.2 (Butantoxin), Potassium channel toxin alpha-KTx 12.3 (Butantoxin- like peptide), Potassium channel toxin alpha-KTx 15.1 (Peptide Aa1), Potassium channel toxin alpha-KTx 15.3 (
  • a CRIP can be a scorpion peptide having an amino acid sequence as set forth in any one of SEQ ID NOs: 88-191.
  • a CRIP can be an imperatoxin. Imperatoxins are peptide toxins derived from the venom of the African scorpion (Pandinus imperator).
  • a CRIP can be an imperatoxin, wherein the imperatoxin is Imperatoxin A (IpTx-a), or a variant thereof.
  • the IpTx- a has an amino acid sequence of GDCLPHLKRCKADNDCCGKKCKRRGTNAEKRCR (SEQ ID NO: 66).
  • a CRIP can be an AaIT1 toxin.
  • the protein toxin, AalT1 is a sodium channel site 4 toxin from North African desert scorpion (Androctonus australis).
  • An exemplary AaIT1 toxin is a peptide having the amino acid sequence according to SEQ ID NO: 88 (NCBI accession No. P01497.2).
  • AaIT1 is a site 4 toxin, which forces the insect sodium channel to open by lowering the activation reaction energy barrier.
  • an scorpion peptide may comprise an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% amino acid sequence identity to SEQ ID NOs: 66, 88-191.
  • a polynucleotide encoding a scorpion peptide or toxin can encode a scorpion peptide or toxin having an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence set forth in SEQ ID NO
  • a CRIP can be isolated from a sea anemone.
  • the sea anemone can be Actinia equina; Anemonia erythraea; Anemonia sulcata; Anemonia viridis; Anthopleura elegantissima; Anthopleura fuscoviridis; Anthopleura xanthogrammica; Bunodosoma caissarum; Bunodosoma cangicum; Bunodosoma granulifera; Heteractis crispa; Parasicyonis actinostoloides; Radianthus paumotensis; or Stoichactis helianthus.
  • the sea anemone toxin can be Av2; an Av3; or a variant thereof.
  • a CRIP can be one of the following sea anemone toxins: Toxin AETX-1 (AETX I), Toxin APETx1, Toxin APETx2, Antihypertensive protein BDS-1 (Blood depressing substance I), Antihypertensive protein BDS-2 (Blood depressing substance II), Neurotoxin Bg-2 (Bg II), Neurotoxin Bg-3 (Bg III), Toxin APE 1-1, Toxin APE 1-2, Neurotoxin-1 (Toxin ATX-I), Neurotoxin-1 (Neurotoxin I), Neurotoxin 1 (Toxin RTX-I), Neurotoxin 1 (Toxin SHP-I), Toxin APE 2-1, Toxin APE 2-2, Neurotoxin-2 (Toxin ATX
  • a CRIP can be a sea anemone peptide having an amino acid sequence as set forth in SEQ ID NOs: 371-411.
  • a CRIP of the present invention can be one or more polypeptides derived from the sea anemone, Anemonia viridis, which possesses a variety of toxins that it uses to defend itself.
  • One of the toxins derived from Anemonia viridis is the neurotoxin “Av3.”
  • Av3 is a type III sea anemone toxin that inhibits the inactivation of voltage-gated sodium (Na + ) channels at receptor site 3, resulting in contractile paralysis.
  • Av3 toxin The binding of an Av3 toxin to site 3 results in the inactivated state of the sodium channel to become destabilized, which in turn causes the channel to remain in the open position (see Blumenthal et al., Voltage-gated sodium channel toxins: poisons, probes, and future promise. Cell Biochem Biophys.2003; 38(2):215-38).
  • Av3 shows high selectivity for crustacean and insect sodium channels, and low selectivity for mammalian sodium channels (see Moran et al., Sea anemone toxins affecting voltage-gated sodium channels - molecular and evolutionary features, Toxicon.2009 Dec 15; 54(8): 1089–1101).
  • a CRIP of the present invention can be an Av3 variant polypeptide (AVP).
  • AVPs can have the following amino acid variations from SEQ ID NO:44: an N-terminal amino acid substitution of R1K relative to SEQ ID NO:44, changing the polypeptide sequence from the wild-type to (SEQ ID NO:45); C-terminal amino acid can be deleted relative to SEQ ID NO:44, changing the polypeptide sequence from the wild- to ID NO:46); and/or an N-terminal mutation and a C-terminal mutation, wherein the N-terminal amino acid can have a substitution of R1K relative to SEQ ID NO:44, and the C-terminal amino acid can be deleted relative to SEQ ID NO:44, changing the polypeptide sequence from the wild-type to (SEQ ID NO:47).
  • an illustrative Av3 peptide or variant thereof is described in the Applicant’s PCT application (Application No. PCT/US19/51093) filed Sept. 13, 2019, entitled “Av3 Mutant Insecticidal Polypeptides and Methods for Producing and Using Same,” the disclosure of which, and the disclosure of Av3 peptides or variants thereof, are described and are incorporated by reference herein in its entirety.
  • a sea anemone peptide may comprise an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% amino acid sequence identity to SEQ ID NOs: 44-47, and 371-411.
  • a polynucleotide encoding a sea anemone peptide can encode a sea anemone peptide having an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence set forth in SEQ ID NOs:
  • Cone shell peptides and conotoxins are toxins isolated from cone shells; these toxins act by interfering with neuronal communication. Examples of conotoxins include the ⁇ -, ⁇ -, ⁇ -, ⁇ -, and ⁇ - conotoxins. Briefly, the ⁇ -conotoxins (and ⁇ A- & ⁇ -conotoxins) target nicotinic ligand gated channels; ⁇ -conotoxins target voltage-gated calcium channels; ⁇ -conotoxins target the voltage-gated sodium channels; ⁇ -conotoxins target the voltage-gated sodium channel; and ⁇ - conotoxins target the voltage-gated potassium channel.
  • a CRIP can be isolated from organisms belonging to the Conus genus, wherein the peptide isolated is a conotoxin.
  • a CRIP can be isolated from Conus amadis; Conus catus; Conus ermineus; Conus geographus; Conus gloriamaris; Conus kinoshitai; Conus magus; Conus marmoreus; Conus purpurascens; Conus stercusmuscarum; Conus striatus; Conus textile; or Conus tulipa.
  • a CRIP can be a toxin, peptide, or protein (otherwise known as a venom- or poison- peptide or protein) that is produced and/or isolated from an arthropod, a spider, a scorpion, an insect, a bee, a wasp, a centipede, a crustacean, a reptile, a snake, a lizard, an amphibian, a frog, a salamander, a mollusk, a cone shell, a cnidarian, a sea anemone, a jellyfish, a hydrozoan, a cephalopod, an octopus, a squid, a cuttlefish, a fish, or a mammal.
  • a CRIP can be a snake venom, or toxin therefrom.
  • CRIP-Insecticidal Proteins are any protein, peptide, polypeptide, amino acid sequence, configuration, or arrangement, consisting of: (1) at least one CRIP, or two or more CRIPs; and (2) additional non-CRIP peptides, polypeptides, or proteins that, e.g., in some embodiments, have the ability to do the following: increase the mortality and/or inhibit the growth of insects when the insects are exposed to a CRIP-insecticidal protein, relative to a CRIP alone; increase the expression of said CRIP-insecticidal protein, e.g., in a host cell or an expression system; and/or affect the post-translational processing of the CRIP-insecticidal protein.
  • a CRIP-insecticidal protein can be a polymer comprising two or more CRIPs.
  • a CRIP-insecticidal protein can be a polymer comprising two or more CRIPs, wherein the CRIPs are operably linked via a linker peptide, e.g., a cleavable and/or non-cleavable linker.
  • a CRIP-insecticidal protein can refer to a one or more CRIPs operably linked with one or more proteins such as a stabilizing domain (STA); an endoplasmic reticulum signaling protein (ERSP); an insect cleavable or insect non-cleavable linker (L); and/or any other combination thereof.
  • a CRIP-insecticidal protein can be a non-naturally occurring protein comprising (1) a wild-type CRIP; and (2) additional peptides, polypeptides, or proteins, e.g., an ERSP; a linker; a STA; a UBI; or a histidine tag or similar marker.
  • a CRIP-insecticidal protein can be a non-naturally occurring protein comprising (1) a wild-type CRIP; and (2) a non-naturally occurring CRIP.
  • a CRIP-insecticidal protein can be a non-naturally occurring protein comprising (1) a wild-type CRIP; and (2) a non-naturally occurring CRIP; and (3) additional peptides, polypeptides, or proteins, e.g., an ERSP; a linker; a STA; a UBI; or a histidine tag or similar marker.
  • a CRIP-insecticidal protein can comprise any of the CRIPs described herein.
  • an insecticidal protein can comprise a one or more CRIPs as disclosed herein.
  • the insecticidal protein can comprise a CRIP homopolymer, e.g., two or more CRIP monomers that are the same CRIP.
  • the insecticidal protein can comprise a CRIP heteropolymer, e.g., two or more CRIP monomers, wherein the CRIP monomers are different.
  • an insecticidal protein can comprise a fused protein comprising two or more CRIPs separated by a cleavable or non-cleavable linker, wherein the amino acid sequence of each CRIP may be the same or different.
  • an insecticidal protein can comprise a fused protein comprising two or more CRIPs separated by a cleavable or non-cleavable linker, wherein the amino acid sequence of each CRIP may be the same or different, wherein the linker is cleavable inside the gut or hemolymph of an insect.
  • an insecticidal protein can comprise a fused protein comprising two or more CRIPs separated by a cleavable or non-cleavable linker, wherein the amino acid sequence of each CRIP may be the same or different, wherein the linker is cleavable inside the gut of a mammal.
  • Exemplary methods for the generation of cleavable and non-cleavable linkers can be found in U.S. Patent Application No.15/727,277; and PCT Application No. PCT/US2013/030042, the disclosure of which are incorporated by reference herein in their entirety.
  • RESULTS FOR PRODUCING A CRIP OR PEPTIDE-IA Methods of producing proteins are well known in the art, and there are a variety of techniques available. For example, in some embodiments, proteins can be produced using recombinant methods, or chemically synthesized. The present disclosure provides methods for producing CRIPs, CRIP-insecticidal proteins, and other peptide insecticidal agents (Peptide-IAs). These methods are described in detail below. [00361] In some embodiments, a CRIP of the present invention can be created using any known method for producing a protein.
  • a CRIP can be created using a recombinant expression system, such as yeast expression system or a bacterial expression system.
  • a recombinant expression system such as yeast expression system or a bacterial expression system.
  • the present invention provides a method of producing a CRIP using a recombinant expression system.
  • the present invention comprises, consists essentially of, or consists of, a method of producing a CRIP, said method comprising: (a) preparing a vector comprising a first expression cassette comprising, consisting essentially of, or consisting of, a polynucleotide operable to encode a CRIP, or a complementary nucleotide sequence thereof, (b) introducing the vector into a host cell, for example a bacteria or a yeast, or an insect, or a plant cell, or an animal cell; and (c) growing the yeast strain in a growth medium under conditions operable to enable expression of the CRIP and secretion into the growth medium.
  • the host cell is a yeast cell.
  • the invention is practicable in a wide variety of host cells (see host cell section below). Indeed, an end-user of the invention can practice the teachings thereof in any host cell of his or her choosing.
  • the host cell can be any host cell that satisfies the requirements of the end-user; i.e., in some embodiments, the expression of a CRIP may be accomplished using a variety of host cells, and pursuant to the teachings herein.
  • a user may desire to use one specific type of host cell (e.g., a yeast cell or a bacteria cell) as opposed to another; the preference of a given host cell can range from availability to cost.
  • the present invention comprises, consists essentially of, or consists of, a method of producing a CRIP, said method comprising: (a) preparing a vector comprising a first expression cassette comprising, consisting essentially of, or consisting of, a polynucleotide operable to encode a CRIP, or a complementary nucleotide sequence thereof; (b) introducing the vector into a host cell, for example a bacteria or a yeast, or an insect, or a plant cell, or an animal cell; and (c) growing the yeast strain in a growth medium under conditions operable to enable expression of the CRIP and secretion into the growth medium.
  • the host cell is a yeast cell.
  • Isolating and mutating wild-type CRIPs [00367]
  • a CRIP or peptide-Insecticidal Agent can be obtained directly from the source (e.g., isolating said CRIP or peptide-IA from an animal).
  • Mutant CRIPs or peptide-IAs can be generated by creating a mutation in the wild-type CRIP or peptide-IA polynucleotide sequence; inserting that CRIP or peptide-IA polynucleotide sequence into the appropriate vector; transforming a host organism in such a way that the polynucleotide encoding a CRIP or peptide-IA is expressed; culturing the host organism to generate the desired amount of CRIP or peptide-IA; and then purifying the CRIP or peptide-IA from in and/or around host organism.
  • Methods of mutagenesis include Kunkel’s method; cassette mutagenesis; PCR site-directed mutagenesis; the “perfect murder” technique (delitto perfetto); direct gene deletion and site-specific mutagenesis with PCR and one recyclable marker; direct gene deletion and site-specific mutagenesis with PCR and one recyclable marker using long homologous regions; transplacement “pop-in pop-out” method; and CRISPR-Cas 9.
  • Exemplary methods of site-directed mutagenesis can be found in Ruvkun & Ausubel, A general method for site-directed mutagenesis in prokaryotes. Nature.1981 Jan 1; 289(5793):85-8; Wallace et al., Oligonucleotide directed mutagenesis of the human beta- globin gene: a general method for producing specific point mutations in cloned DNA. Nucleic Acids Res.1981 Aug 11; 9(15):3647-56; Dalbadie-McFarland et al., Oligonucleotide-directed mutagenesis as a general and powerful method for studies of protein function.
  • Wild-type CRIPs e.g., spider, scorpion, and/or other toxins can be isolated from the venom.
  • spider venom can be isolated from the venom glands of spiders (e.g., spiders such as Eratigena agrestis), using any of the techniques known to those having ordinary skill in the art.
  • spiders e.g., spiders such as Eratigena agrestis
  • venom can be isolated from spiders according to the methods described in U.S. Patent No 5,688,764, the disclosure of which is incorporated herein by reference in its entirety.
  • a wild-type CRIP or peptide-IA polynucleotide sequence can be obtained by screening a genomic library using primer probes directed to the CRIP or peptide-IA polynucleotide sequence.
  • wild-type CRIP or peptide-IA polynucleotide sequence and/or mutant CRIP or peptide-IA polynucleotide sequences can be chemically synthesized.
  • a CRIP or peptide-IA polynucleotide sequence and/or mutant CRIP or peptide-IA polynucleotide sequence can be generated using the oligonucleotide synthesis methods such as the phosphoramidite; triester, phosphite, or H-Phosphonate methods. See Engels, J. W. and Uhlmann, E. (1989), Gene Synthesis (New Synthetic Methods (77)). Angew. Chem. Int. Ed. Engl., 28: 716–734, the disclosure of which is incorporated herein by reference in its entirety.
  • the polynucleotide sequence encoding the CRIP or peptide-IA can be chemically synthesized using commercially available polynucleotide synthesis services such as those offered by GENEWIZ® (e.g., TurboGENE TM ; PriorityGENE; and FragmentGENE), or SIGMA-ALDRICH® (e.g., Custom DNA and RNA Oligos Design and Order Custom DNA Oligos).
  • GENEWIZ® e.g., TurboGENE TM ; PriorityGENE; and FragmentGENE
  • SIGMA-ALDRICH® e.g., Custom DNA and RNA Oligos Design and Order Custom DNA Oligos.
  • Exemplary method for generating DNA and or custom chemically synthesized polynucleotides are well known in the art, and are illustratively provided in U.S.
  • Chemically synthesizing polynucleotides allows for a DNA sequence to be generated that is tailored to produce a desired polypeptide based on the arrangement of nucleotides within said sequence (i.e., the arrangement of cytosine [C], guanine [G], adenine [A] or thymine [T] molecules); the mRNA sequence that is transcribed from the chemically synthesized DNA polynucleotide can be translated to a sequence of amino acids, each amino acid corresponding to a codon in the mRNA sequence.
  • the amino acid composition of a polypeptide chain that is translated from an mRNA sequence can be altered by changing the underlying codon that determines which of the 20 amino acids will be added to the growing polypeptide; thus, mutations in the DNA such as insertions, substitutions, deletions, and frameshifts may cause amino acid insertions, substitutions, or deletions, depending on the underlying codon.
  • Obtaining a CRIP or peptide-IA from a chemically synthesized DNA polynucleotide sequence and/or a wild-type DNA polynucleotide sequence that has been altered via mutagenesis can be achieved by cloning the DNA sequence into an appropriate vector.
  • the vector can be a plasmid, which can introduce a heterologous gene and/or expression cassette into yeast cells to be transcribed and translated.
  • the term “vector” is used to refer to a carrier nucleic acid molecule into which a nucleic acid sequence can be inserted for introduction into a cell where it can be replicated.
  • a vector may contain “vector elements” such as an origin of replication (ORI); a gene that confers antibiotic resistance to allow for selection; multiple cloning sites; a promoter region; a selection marker for non-bacterial transfection; and a primer binding site.
  • ORI origin of replication
  • a nucleic acid sequence can be “exogenous,” which means that it is foreign to the cell into which the vector is being introduced or that the sequence is homologous to a sequence in the cell but in a position within the host cell nucleic acid in which the sequence is ordinarily not found.
  • Vectors include plasmids, cosmids, viruses (bacteriophage, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs).
  • plasmids include plasmids, cosmids, viruses (bacteriophage, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs).
  • a vector may encode a targeting molecule.
  • a targeting molecule is one that directs the desired nucleic acid to a particular tissue, cell, or other location.
  • vectors and transformation [00376]
  • a CRIP or peptide-IA polynucleotide can be cloned into a vector using a variety of cloning strategies, and commercial cloning kits and materials readily available to those having ordinary skill in the art.
  • the CRIP or peptide- IA polynucleotide can be cloned into a vector using such strategies as the SnapFast; Gateway; TOPO; Gibson; LIC; InFusionHD; or Electra strategies.
  • SnapFast Gateway
  • TOPO TOPO
  • Gibson Gibson
  • LIC InFusionHD
  • Electra Electra strategies
  • a CRIP or peptide-IA polynucleotide can be generated using polymerase chain reaction (PCR), and combined with a pCR TM II-TOPO vector, or a PCR TM 2.1-TOPO® vector (commercially available as the TOPO® TA Cloning ® Kit from Invitrogen) for 5 minutes at room temperature; the TOPO® reaction can then be transformed into competent cells, which can subsequently be selected based on color change (see Janke et al., A versatile toolbox for PCR-based tagging of yeast genes: new fluorescent proteins, more markers and promoter substitution cassettes. Yeast.2004 Aug; 21(11):947-62; see also, Adams et al. Methods in Yeast Genetics.
  • PCR polymerase chain reaction
  • a polynucleotide encoding a CRIP or peptide-IA can be cloned into a vector such as a plasmid, cosmid, virus (bacteriophage, animal viruses, and plant viruses), and/or artificial chromosome (e.g., YACs).
  • a polynucleotide encoding a CRIP or peptide-IA can be inserted into a vector, for example, a plasmid vector using E.
  • coli as a host, by performing the following: digesting about 2 to 5 ⁇ g of vector DNA using the restriction enzymes necessary to allow the DNA segment of interest to be inserted, followed by overnight incubation to accomplish complete digestion (alkaline phosphatase may be used to dephosphorylate the 5’- end in order to avoid self-ligation/recircularization); gel purify the digested vector.
  • amplify the DNA segment of interest for example, a polynucleotide encoding an CRIP or peptide-IA, via PCR, and remove any excess enzymes, primers, unincorporated dNTPs, short-failed PCR products, and/or salts from the PCR reaction using techniques known to those having ordinary skill in the art (e.g., by using a PCR clean-up kit).
  • Ligate the DNA segment of interest to the vector by creating a mixture comprising: about 20 ng of vector; about 100 to 1,000 ng or DNA segment of interest; 2 ⁇ L 10x buffer (i.e., 30 mM Tris-HCl 4 mM MgCl 2 , 26 ⁇ M NAD, 1 mM DTT, 50 ⁇ g/ml BSA, pH 8, stored at 25°C); 1 ⁇ L T4 DNA ligase; all brought to a total volume of 20 ⁇ L by adding H 2 O.
  • the ligation reaction mixture can then be incubated at room temperature for 2 hours, or at 16°C for an overnight incubation.
  • the ligation reaction (i.e., about 1 ⁇ L) can then be transformed to competent cell, for example, by using electroporation or chemical methods, and a colony PCR can then be performed to identify vectors containing the DNA segment of interest.
  • a polynucleotide encoding a CRIP or peptide-IA, along with other DNA segments together composing a CRIP or peptide-IA expression ORF can be designed for secretion from host yeast cells.
  • an illustrative method of designing a CRIP or peptide-IA expression ORF is as follows: the ORF can begin with a signal peptide sequence, followed by a DNA sequence encoding a Kex2 cleavage site (Lysine-Arginine), and subsequently followed by the CRIP or peptide-IA polynucleotide transgene, with the addition of glycine-serine codons at the 5’-end, and finally a stop codon at the 3’-end. All these elements will then be expressed to a fusion peptide in yeast cells as a single open reading frame (ORF).
  • ⁇ -mating factor ( ⁇ MF) signal sequence is most frequently used to facilitate metabolic processing of the recombinant insecticidal peptides through the endogenous secretion pathway of the recombinant yeast, i.e. the expressed fusion peptide will typically enter the Endoplasmic Reticulum, wherein the ⁇ -mating factor signal sequence is removed by signal peptidase activity, and then the resulting pro-insecticidal peptide will be trafficked to the Golgi Apparatus, in which the Lysine-Arginine dipeptide mentioned above is completely removed by Kex2 endoprotease, after which the mature, polypeptide (i.e., CRIP or peptide-IA), is secreted out of the cells.
  • ⁇ MF ⁇ -mating factor
  • polypeptide expression levels in recombinant yeast cells can be enhanced by optimizing the codons based on the specific host yeast species.
  • Naturally occurring frequencies of codons observed in endogenous open reading frames of a given host organism need not necessarily be optimized for high efficiency expression.
  • different yeast species for example, Kluyveromyces lactis, Pichia pastoris, Saccharomyces cerevisiae, etc.
  • a codon-optimized CRIP or peptide-IA expression ORF can be ligated into a yeast-specific expression vectors for yeast expression.
  • yeast-specific expression vectors for yeast expression.
  • yeast expression There are many expression vectors available for yeast expression, including episomal vectors and integrative vectors, and they are usually designed for specific yeast strains. One should carefully choose the appropriate expression vector in view of the specific yeast expression system which will be used for the peptide production.
  • integrative vectors can be used, which integrate into chromosomes of the transformed yeast cells and remain stable through cycles of cell division and proliferation.
  • the integrative DNA sequences are homologous to targeted genomic DNA loci in the transformed yeast species, and such integrative sequences include pLAC4, 25S rDNA, pAOX1, and TRP2, etc.
  • the locations of insecticidal peptide transgenes can be adjacent to the integrative DNA sequence (Insertion vectors) or within the integrative DNA sequence (replacement vectors).
  • the expression vectors can contain E. coli elements for DNA preparation in E. coli, for example, E. coli replication origin, antibiotic selection marker, etc.
  • vectors can contain an array of the sequence elements needed for expression of the transgene of interest, for example, transcriptional promoters, terminators, yeast selection markers, integrative DNA sequences homologous to host yeast DNA, etc.
  • yeast promoters include natural and engineered promoters, for example, yeast promoters such as pLAC4, pAOX1, pUPP, pADH1, pTEF, pGal1, etc., and others, can be used in some embodiments.
  • selection methods such as acetamide prototrophy selection; zeocin-resistance selection; geneticin-resistance selection; nourseothricin- resistance selection; uracil deficiency selection; and/or other selection methods may be used.
  • the Aspergillus nidulans amdS gene can be used as selectable marker. Exemplary methods for the use of selectable markers can be found in U.S. Patent Nos.6,548,285 (filed Apr.3, 1997); 6,165,715 (filed June 22, 1998); and 6,110,707 (filed Jan.17, 1997), the disclosures of which are incorporated herein by reference in its entirety.
  • a polynucleotide encoding a CRIP or peptide-IA can be inserted into a pKLAC1 plasmid.
  • the pKLAC1 is commercially available from New England Biolabs® Inc., (item no. (NEB #E1000).
  • the pKLAC1 is designed to accomplish high-level expression of recombinant protein (e.g., CRIP or peptide-IA) in the yeast Kluyveromyces lactis.
  • the pKLAC1 plasmid can be ordered alone, or as part of a K. lactis Protein Expression Kit.
  • the pKLAC1 plasmid can be linearized using the SacII or BstXI restriction enzymes, and possesses a MCS downstream of an ⁇ MF secretion signal.
  • the ⁇ MF secretion signal directs recombinant proteins to the secretory pathway, which is then subsequently cleaved via Kex2 resulting in, for example, a CRIP or peptide-IA.
  • Kex2 is a calcium-dependent serine protease, which is involved in activating proproteins of the secretory pathway, and is commercially available (PeproTech®; item no.450-45).
  • a polynucleotide encoding a CRIP or peptide-IA can be inserted into a pKlac1 plasmid, or subcloned into a pKlac1 plasmid subsequent to selection of yeast colonies transformed with pKLAC1 plasmids ligated with polynucleotide encoding a CRIP or peptide-IA.
  • yeast for example K.
  • lactis transformed with a pKLAC1 plasmids ligated with polynucleotide encoding a CRIP or peptide-IA can be selected based on acetamidase (amdS), which allows transformed yeast cells to grow in YCB medium containing acetamide as its only nitrogen source. Once positive yeast colonies transformed with a pKLAC1 plasmids ligated with polynucleotide encoding a CRIP or peptide-IA are identified.
  • amdS acetamidase
  • a polynucleotide encoding a CRIP or peptide-IA can be inserted into other commercially available plasmids and/or vectors that are readily available to those having skill in the art, e.g., plasmids are available from Addgene (a non-profit plasmid repository); GenScript®; Takara®; Qiagen®; and Promega TM .
  • a polynucleotide encoding a TVP can be inserted into other commercially available plasmids and/or vectors that are readily available to those having skill in the art, e.g., plasmids are available from Addgene (a non-profit plasmid repository); GenScript®; Takara®; Qiagen®; and Promega TM .
  • a yeast cell transformed with one or more CRIP expression cassettes can produce a CRIP in a yeast culture with a yield of: at least 70 mg/L, at least 80 mg/L, at least 90 mg/L, at least 100 mg/L, at least 110 mg/L, at least 120 mg/L, at least 130 mg/L, at least 140 mg/L, at least 150 mg/L, at least 160 mg/L, at least 170 mg/L, at least 180 mg/L, at least 190 mg/L 200 mg/L, at least 500 mg/L, at least 750 mg/L, at least 1,000 mg/L, at least 1,250 mg/L, at least 1,500 mg/L, at least 1,750 mg/L, at least 2,000 mg/L, at least 2,500 mg/L, at least 3,000 mg/L, at least 3,500 mg/L, at least 4,000 mg/L, at least 4,500 mg/L, at least 5,000 mg/L, at least 5,500 mg/L, at least at least 6,000 mg
  • one or more expression cassettes comprising a polynucleotide operable to express a CRIP can be inserted into a vector, resulting in a yield ranging from about 100 mg/L of CRIP to about 100,000 mg/L; from about 110 mg/L to about 100,000 mg/L; from about 120 mg/L to about 100,000 mg/L; from about 130 mg/L to about 100,000 mg/L; from about 140 mg/L to about 100,000 mg/L; from about 150 mg/L to about 100,000 mg/L; from about 160 mg/L to about 100,000 mg/L; from about 170 mg/L to about 100,000 mg/L; from about 180 mg/L to about 100,000 mg/L; from about 190 mg/L to about 100,000 mg/L; from about 200 mg/L to about 100,000 mg/L; from about 250 mg/L to about 100,000 mg/L; from about 500 mg/L to about 100,000 mg/L; from about 750 mg/L to about 100,000 mg/L; from about 1000 mg/L to about 100,000 mg/L; from about
  • one or more expression cassettes comprising a polynucleotide operable to express a CRIP can be inserted into a vector, resulting in a yield ranging from about 100 mg/L of CRIP to about 100,000 mg/L; from about 100 mg/L to about 99500 mg/L; from about 100 mg/L to about 99000 mg/L; from about 100 mg/L to about 98500 mg/L; from about 100 mg/L to about 98000 mg/L; from about 100 mg/L to about 97500 mg/L; from about 100 mg/L to about 97000 mg/L; from about 100 mg/L to about 96500 mg/L; from about 100 mg/L to about 96000 mg/L; from about 100 mg/L to about 95500 mg/L; from about 100 mg/L to about 95000 mg/L; from about 100 mg/L to about 94500 mg/L; from about 100 mg/L to about 94000 mg/L; from about 100 mg/L to about 93500 mg
  • additional DNA segments known as regulatory elements can be cloned into a vector that allow for enhanced expression of the foreign DNA or transgene; examples of such additional DNA segments include (1) promoters, terminators, and/or enhancer elements; (2) an appropriate mRNA stabilizing polyadenylation signal; (3) an internal ribosome entry site (IRES); (4) introns; and (5) post-transcriptional regulatory elements.
  • additional DNA segments include (1) promoters, terminators, and/or enhancer elements; (2) an appropriate mRNA stabilizing polyadenylation signal; (3) an internal ribosome entry site (IRES); (4) introns; and (5) post-transcriptional regulatory elements.
  • a single expression cassette can contain one or more of the aforementioned regulatory elements, and a polynucleotide operable to express a CRIP or peptide-IA.
  • a CRIP or peptide-IA expression cassette can comprise polynucleotide operable to express a CRIP or peptide-IA, and an ⁇ -MF signal; Kex2 site; LAC4 terminator; ADN1 promoter; and an acetamidase (amdS) selection marker—flanked by LAC4 promoters on the 5’-end and 3’-end.
  • there can be a first expression cassette comprising a polynucleotide operable to express a CRIP or peptide-IA.
  • there are two expression cassettes operable to encode a CRIP or peptide-IA i.e., a double expression cassette.
  • there are three expression cassettes operable to encode a CRIP or peptide-IA i.e., a triple expression cassette).
  • a double expression cassette can be generated by subcloning a second CRIP or peptide-IA expression cassette into a vector containing a first CRIP or peptide-IA expression cassette.
  • a triple expression cassette can be generated by subcloning a third CRIP or peptide-IA expression cassette into a vector containing a first and a second CRIP or peptide-IA expression cassette.
  • a yeast cell transformed with one or more CRIP or peptide-IA expression cassettes can produce CRIP or peptide-IA in a yeast culture with a yield of: at least 70 mg/L, at least 80 mg/L, at least 90 mg/L, at least 100 mg/L, at least 110 mg/L, at least 120 mg/L, at least 130 mg/L, at least 140 mg/L, at least 150 mg/L, at least 160 mg/L, at least 170 mg/L, at least 180 mg/L, at least 190 mg/L 200 mg/L, at least 500 mg/L, at least 750 mg/L, at least 1,000 mg/L, at least 1,250 mg/L, at least 1,500 mg/L, at least 1,750 mg/L, at least 2,000 mg/L, at least 2,500 mg/L, at least 3,000 mg/L, at least 3,500 mg/L, at least 4,000 mg/L, at least 4,500 mg/L, at least 5,000 mg/L, at least 5,
  • one or more expression cassettes comprising a polynucleotide operable to express a CRIP or peptide-IA can be inserted into a vector, for example a pKlac1 plasmid, resulting in a yield of about 100 mg/L of CRIP or peptide-IA (supernatant of yeast fermentation broth).
  • a vector for example a pKlac1 plasmid
  • two expression cassettes comprising a polynucleotide operable to express a CRIP or peptide-IA can be inserted into a vector, for example a pKS482 plasmid, resulting in a yield of about 2 g/L of CRIP or peptide-IA (supernatant of yeast fermentation broth).
  • three expression cassettes comprising a polynucleotide operable to express a CRIP or peptide-IA can be inserted into a vector, for example a pKlac1T plasmid.
  • a vector for example a pKlac1T plasmid.
  • multiple CRIP or peptide-IA expression cassettes can be transfected into yeast in order to enable integration of one or more copies of the optimized CRIP or peptide-IA transgene into the K. lactis genome.
  • lactis genome is as follows: a CRIP or peptide-IA expression cassette DNA sequence is synthesized, comprising an intact LAC4 promoter element, a codon-optimized CRIP or peptide-IA expression ORF element and a pLAC4 terminator element; the intact expression cassette is ligated into the pKlac1 vector between Sal I and Kpn I restriction sites, downstream of the pLAC4 terminator of pKS477, resulting in the double transgene CRIP or peptide-IA expression vector, pKS482; the double transgene vectors, pKS482, are then linearized using Sac II restriction endonuclease and transformed into YCT306 strain of K. lactis by electroporation.
  • the resulting yeast colonies are then grown on YCB agar plate supplemented with 5 mM acetamide, which only the acetamidase-expressing cells could use efficiently as a metabolic source of nitrogen.
  • agar plate supplemented with 5 mM acetamide, which only the acetamidase-expressing cells could use efficiently as a metabolic source of nitrogen.
  • about 100 to 400 colonies can be picked from the pKS482 yeast plates. Inoculates from the colonies are each cultured in 2.2 mL of the defined K. lactis media with 2% sugar alcohol added as a carbon source. Cultures are incubated at 23.5°C, with shaking at 280 rpm, for six days, at which point cell densities in the cultures will reach their maximum levels as indicated by light absorbance at 600 nm (OD600).
  • chemical peptide synthesis can be achieved using Liquid phase peptide synthesis (LPPS), or solid phase peptide synthesis (SPPS).
  • LPPS Liquid phase peptide synthesis
  • SPPS solid phase peptide synthesis
  • peptide synthesis can generally be achieved by using a strategy wherein the coupling the carboxyl group of a subsequent amino acid to the N- terminus of a preceding amino acid generates the nascent polypeptide chain—a process that is opposite to the type of polypeptide synthesis that occurs in nature.
  • Peptide deprotection is an important first step in the chemical synthesis of polypeptides.
  • Peptide deprotection is the process in which the reactive groups of amino acids are blocked through the use of chemicals in order to prevent said amino acid’s functional group from taking part in an unwanted or non-specific reaction or side reaction; in other words, the amino acids are “protected” from taking part in these undesirable reactions.
  • the amino acids Prior to synthesizing the peptide chain, the amino acids must be “deprotected” to allow the chain to form (i.e., amino acids to bind).
  • Chemicals used to protect the N-termini include 9-fluorenylmethoxycarbonyl (Fmoc), and tert-butoxycarbonyl (Boc), each of which can be removed via the use of a mild base (e.g., piperidine) and a moderately strong acid (e.g., trifluoracetic acid (TFA)), respectively.
  • a mild base e.g., piperidine
  • a moderately strong acid e.g., trifluoracetic acid (TFA)
  • TFA trifluoracetic acid
  • the incoming amino acid’s C-terminal carboxylic acid must be activated: this can be accomplished using carbodiimides such as diisopropylcarbodiimide (DIC), or dicyclohexylcarbodiimide (DCC), which react with the incoming amino acid’s carboxyl group to form an O-acylisourea intermediate.
  • the O-acylisourea intermediate is subsequently displaced via nucleophilic attack via the primary amino group on the N- terminus of the growing peptide chain.
  • the reactive intermediate generated by carbodiimides can result in the racemization of amino acids.
  • reagents such as 1-hydroxybenzotriazole (HOBt) are added in order to react with the O- acylisourea intermediate.
  • HOBt 1-hydroxybenzotriazole
  • Other couple agents include 2-(1H-benzotriazol-1- yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), and benzotriazol-1-yl-oxy- tris(dimethylamino)phosphonium hexafluorophosphate (BOP), with the additional activating bases.
  • peptides can be purified based on the peptide’s physiochemical characteristics (e.g., charge, size, hydrophobicity, etc.).
  • Techniques that can be used to purify peptides include Purification techniques include Reverse-phase chromatography (RPC); Size-exclusion chromatography; Partition chromatography; High- performance liquid chromatography (HPLC); and Ion exchange chromatography (IEC).
  • RPC Reverse-phase chromatography
  • Size-exclusion chromatography Size-exclusion chromatography
  • Partition chromatography Partition chromatography
  • HPLC High- performance liquid chromatography
  • IEC Ion exchange chromatography
  • transformation to describe processes where exogenous and/or heterologous DNA or RNA are introduced into a bacterial cell; and reserve the term “transfection” for processes that describe the introduction of exogenous and/or heterologous DNA or RNA into eukaryotic cells.
  • transformation and “transfection” are used synonymously, regardless of whether a process describes the introduction exogenous and/or heterologous DNA or RNA into a prokaryote (e.g., bacteria) or a eukaryote (e.g., yeast, plants, or animals).
  • a host cell can be transformed using the following methods: electroporation; cell squeezing; microinjection; impalefection; the use of hydrostatic pressure; sonoporation; optical transfection; continuous infusion; lipofection; through the use of viruses such as adenovirus, adeno-associated virus, lentivirus, herpes simplex virus, and retrovirus; the chemical phosphate method; endocytosis via DEAE- dextran or polyethylenimine (PEI); protoplast fusion; hydrodynamic deliver; magnetofection; nucleoinfection; and/or others.
  • viruses such as adenovirus, adeno-associated virus, lentivirus, herpes simplex virus, and retrovirus
  • viruses such as adenovirus, adeno-associated virus, lentivirus, herpes simplex virus, and retrovirus
  • viruses such as adenovirus, adeno-associated virus, lentivirus, herpes simplex virus, and retrovirus
  • viruses
  • Electroporation is a technique in which electricity is applied to cells causing the cell membrane to become permeable; this in turn allows exogenous DNA to be introduced into the cells. Electroporation is readily known to those having ordinary skill in the art, and the tools and devices required to achieve electroporation are commercially available (e.g., Gene Pulser XcellTM Electroporation Systems, Bio-Rad®; Neon® Transfection System for Electroporation, Thermo-Fisher Scientific; and other tools and/or devices). Exemplary methods of electroporation are illustrated in Potter & Heller, Transfection by Electroporation.
  • electroporation can be used to introduce a vector containing a polynucleotide encoding a CRIP or peptide-IA into yeast, for example, a CRIP or peptide-IA cloned into a pKlac1 plasmid, and transformed into K.
  • lactis cells via electroporation can be accomplished by inoculating about 10-200 mL of yeast extract peptone dextrose (YEPD) with a suitable yeast species, for example, Kluyveromyces lactis, Kluyveromyces marxianus, Saccharomyces cerevisiae, Pichia pastoris, etc., and incubate on a shaker at 30°C until the early exponential phase of yeast culture (e.g.
  • galactose, maltose, latotriose, sucrose, fructose or glucose and/or sugar alcohol for example, erythritol, hydrogenated starch hydrolysates, isomalt, lactitol, maltitol, mannitol, and xylitol, followed by spinning down at 3,000 rpm for 5 minutes; resuspending the cells with proper volume of ice cold 1M fermentable sugar, e.g.
  • galactose maltose, latotriose, sucrose, fructose or glucose and/or a sugar alcohol, for example, erythritol, hydrogenated starch hydrolysates, isomalt, lactitol, maltitol, mannitol, and xylitol mixture, and then spreading onto selective plates.
  • a sugar alcohol for example, erythritol, hydrogenated starch hydrolysates, isomalt, lactitol, maltitol, mannitol, and xylitol mixture, and then spreading onto selective plates.
  • electroporation can be used to introduce a vector containing a polynucleotide encoding a CRIP or peptide-IA into plant protoplasts by incubating sterile plant material in a protoplast solution (e.g., around 8 mL of 10 mM 2-[N- morpholino]ethanesulfonic acid (MES), pH 5.5; 0.01% (w/v) pectylase; 1% (w/v) macerozyme; 40 mM CaCl 2 ; and 0.4 M mannitol) and adding the mixture to a rotary shaker for about 3 to 6 hours at 30°C to produce protoplasts; removing debris via 80- ⁇ m-mesh nylon screen filtration; rinsing the screen with about 4 ml plant electroporation buffer (e.g., 5 mM CaCl 2 ; 0.4 M mannitol; and PBS); combining the protoplasts in a protoplast solution
  • MES 2-[N
  • Host Cells may be implemented in any cell type, e.g., a eukaryotic or prokaryotic cell.
  • the host cell used to produce a CRIP, a CRIP- insecticidal protein, or a peptide-IA is a prokaryote.
  • the host cell may be an Archaebacteria or Eubacteria, such as Gram-negative or Gram-positive organisms. Examples of useful bacteria include Escherichia (e.g., E. coli), Bacilli (e.g., B.
  • the host cell used to produce a CRIP, a CRIP- insecticidal protein, or a peptide-IA may be a unicellular cell.
  • the host cell may be bacterial cells such as gram positive bacteria.
  • the host cell may be a bacteria selected from the following genera consisting of: Candidatus Chloracidobacterium, Arthrobacter, Corynebacterium, Frankia, Micrococcus, Mycobacterium, Propionibacterium, Streptomyces, Aquifex Bacteroides, Porphyromonas, Bacteroides, Porphyromonas, Flavobacterium, Chlamydia, Prosthecobacter, Verrucomicrobium, Chloroflexus, Chroococcus, Merismopedia, Synechococcus, Anabaena, Nostoc, Spirulina, Trichodesmium, Pleurocapsa, Prochlorococcus, Prochloron, Bacillus, Listeria, Staphylococcus, Clostridium, Dehalobacter, Epulopiscium, Ruminococcus, Enterococcus, Lactobacillus, Streptococcus, Erysipel
  • the host cell used to produce a CRIP, a CRIP- insecticidal protein, or a peptide-IA may be selected from one of the following bacteria species: Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus coagulans, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus stearothermophilus, Bacillus subtilis, Bacillus thuringiensis, Streptomyces lividans, Streptomyces murinus, Streptomyces coelicolor, Streptomyces albicans, Streptomyces griseus, Streptomyces plicatosporus, Escherichia albertii, Escherichia blattae, Escherichia coli, Escherichia fergusonii, Escherichi
  • the host cell used to produce a CRIP, a CRIP- insecticidal protein, or a peptide-IA can be eukaryote.
  • the host cell used to produce a CRIP, a CRIP- insecticidal protein, or a peptide-IA may be a cell belonging to the clades: Opisthokonta; Viridiplantae (e.g., algae and plant); Amebozoa; Cercozoa; Alveolata; Marine flagellates; Heterokonta; Discicristata; or Excavata.
  • the procedures and methods described here can be accomplished using a host cell that is, e.g., a Metazoan, a Choanoflagellata, or a fungi.
  • the procedures and methods described here can be accomplished using a host cell that is a fungi.
  • the host cell may be a cell belonging to the eukaryote phyla: Ascomycota, Basidiomycota, Chytridiomycota, Microsporidia, or Zygomycota
  • the procedures and methods described here can be accomplished using a host cell that is a fungi belonging to one of the following genera: Aspergillus, Cladosporium, Magnaporthe, Morchella, Neurospora, Penicillium, Saccharomyces, Cryptococcus, or Ustilago.
  • the procedures and methods described here can be accomplished using a host cell that is a fungi belonging to one of the following species: Saccharomyces cerevisiae, Saccharomyces boulardi, Saccharomyces uvarum; Aspergillus flavus, A. terreus, A. awamori; Cladosporium elatum, Cl. Herbarum, Cl. Sphaerospermum, and Cl.
  • the procedures and methods described here can be accomplished using a host cell that is a Kluyveromyces lactis, Kluyveromyces marxianus, Saccharomyces cerevisiae, or Pichia pastoris.
  • the host cell used to produce a CRIP, a CRIP- insecticidal protein, or a peptide-IA may be a fungi belonging to one of the following genera: Aspergillus, Cladosporium, Magnaporthe, Morchella, Neurospora, Penicillium, Saccharomyces, Cryptococcus, or Ustilago.
  • the host cell used to produce a CRIP, a CRIP- insecticidal protein, or a peptide-IA may be a member of the Saccharomycetaceae family.
  • the host cell may be one of the following genera within the Saccharomycetaceae family: Brettanomyces, Candida, Citeromyces, Cyniclomyces, Debaryomyces, Issatchenkia, Kazachstania, Kluyveromyces, Komagataella, Kuraishia, Lachancea, Lodderomyces, Nakaseomyces, Pachysolen, Pichia, Saccharomyces, Spathaspora, Tetrapisispora, Vanderwaltozyma, Torulaspora, Williopsis, Zygosaccharomyces, or Zygotorulaspora.
  • the host cell used to produce a CRIP, a CRIP- insecticidal protein, or a peptide-IA may be one of the following: Aspergillus flavus, Aspergillus terreus, Aspergillus awamori, Cladosporium elatum, Cladosporium Herbarum, Cladosporium Sphaerospermum, Cladosporium cladosporioides, Magnaporthe grisea, Magnaporthe oryzae, Magnaporthe rhizophila, Morchella deliciosa, Morchella esculenta, Morchella conica, Neurospora crassa, Neurospora intermedia, Neurospora tetrasperma, Penicillium notatum, Penicillium chrysogenum, Penicillium roquefortii, or Penicillium simplicissimum.
  • the host cell used to produce a CRIP, a CRIP- insecticidal protein, or a peptide-IA may be a species within the Candida genus.
  • the host cell may be one of the following: Candida albicans, Candida ascalaphidarum, Candida amphixiae, Candida antarctica, Candida argentea, Candida atlantica, Candida atmosphaerica, Candida auris, Candida blankii, Candida blattae, Candida bracarensis, Candida bromeliacearum, Candida carpophila, Candida carvajalis, Candida cerambycidarum, Candida chauliodes, Candida corydalis, Candida dosseyi, Candida dubliniensis, Candida ergatensis, Candida fructus, Candida glabrata, Candida fermentati, Candida guilliermondii, Candida haemulonii, Candida humilis, Candida insectamens, Candida insectorum, Candida intermedia, Candida jef
  • the host cell used to produce a CRIP, a CRIP- insecticidal protein, or a peptide-IA may be a species within the Kluyveromyces genus.
  • the host cell may be one of the following: Kluyveromyces aestuarii, Kluyveromyces dobzhanskii, Kluyveromyces lactis, Kluyveromyces marxianus, Kluyveromyces nonfermentans, or Kluyveromyces wickerhamii.
  • the host cell used to produce a CRIP, a CRIP- insecticidal protein, or a peptide-IA may be a species within the Pichia genus.
  • the host cell may be one of the following: Pichia farinose, Pichia anomala, Pichia heedii, Pichia guilliermondii, Pichia kluyveri, Pichia membranifaciens, Pichia norvegensis, Pichia ohmeri, Pichia pastoris, Pichia methanolica, or Pichia subpelliculosa.
  • the host cell used to produce a CRIP, a CRIP- insecticidal protein, or a peptide-IA may be a species within the Saccharomyces genus.
  • the host cell may be one of the following: Saccharomyces arboricolus, Saccharomyces bayanus, Saccharomyces bulderi, Saccharomyces cariocanus, Saccharomyces cariocus, Saccharomyces cerevisiae, Saccharomyces cerevisiae var boulardii, Saccharomyces chevalieri, Saccharomyces dairenensis, Saccharomyces ellipsoideus, Saccharomyces eubayanus, Saccharomyces exiguous, Saccharomyces florentinus, Saccharomyces fragilis, Saccharomyces kudriavzevii, Saccharomyces martiniae, Saccharomyces mi
  • the host cell used to produce a CRIP, a CRIP- insecticidal protein, or a peptide-IA may be one of the following: Saccharomyces cerevisiae, Pichia pastoris, Pichia methanolica, Schizosaccharomyces pombe, or Hansenula anomala.
  • Saccharomyces cerevisiae Pichia pastoris
  • Pichia methanolica Pichia methanolica
  • Schizosaccharomyces pombe or Hansenula anomala.
  • the use of yeast cells as a host organism to generate recombinant CRIPs or peptide-IAs is an exceptional method, well known to those having ordinary skill in the art.
  • the methods and compositions described herein can be performed with any species of yeast, including but not limited to any species of the genus Saccharomyces, Pichia, Kluyveromyces, Hansenula, Yarrowia or Schizosaccharomyces and the species Saccharomyces includes any species of Saccharomyces, for example Saccharomyces cerevisiae species selected from following strains: INVSc1, YNN27, S150-2B, W303-1B, CG25, W3124, JRY188, BJ5464, AH22, GRF18, W303-1A and BJ3505.
  • members of the Pichia species including any species of Pichia for example the Pichia species, Pichia pastoris, for example, the Pichia pastoris is selected from following strains: Bg08, Y-11430, X-33, GS115, GS190, JC220, JC254, GS200, JC227, JC300, JC301, JC302, JC303, JC304, JC305, JC306, JC307, JC308, YJN165, KM71, MC100-3, SMD1163, SMD1165, SMD1168, GS241, MS105, any pep4 knock-out strain and any prb1 knock-out strain, as well as Pichia pastoris selected from following strains: Bg08, X-33, SMD1168 and KM71.
  • any Kluyveromyces species can be used to accomplish the methods described here, including any species of Kluyveromyces, for example, Kluyveromyces lactis, and we teach that the stain of Kluyveromyces lactis can be but is not required to be selected from following strains: GG799, YCT306, YCT284, YCT389, YCT390, YCT569, YCT598, NRRL Y-1140, MW98-8C, MS1, CBS293.91, Y721, MD2/1, PM6-7A, WM37, K6, K7, 22AR1, 22A295-1, SD11, MG1/2, MSK110, JA6, CMK5, HP101, HP108 and PM6-3C, in addition to Kluyveromyces lactis species is selected from GG799, YCT306 and NRRL Y-1140.
  • the host cell used to produce a CRIP, a CRIP- insecticidal protein, or a peptide-IA can be an Aspergillus oryzae.
  • the host cell used to produce a CRIP, a CRIP- insecticidal protein, or a peptide-IA can be an Aspergillus japonicas.
  • the host cell used to produce a CRIP, a CRIP- insecticidal protein, or a peptide-IA can be an Aspergillus niger.
  • the host cell used to produce a CRIP, a CRIP- insecticidal protein, or a peptide-IA can be a Bacillus licheniformis.
  • the host cell used to produce a CRIP, a CRIP- insecticidal protein, or a peptide-IA can be a Bacillus subtilis.
  • the host cell used to produce a CRIP, a CRIP- insecticidal protein, or a peptide-IA can be a Trichoderma reesei.
  • the procedures and methods described here can be accomplished using a host cell that is a yeast, including but not limited to any species of Hansenula species including any species of Hansenula and preferably Hansenula polymorpha.
  • the procedures and methods described here can be accomplished with any species of yeast, including but not limited to any species of Yarrowia species for example, Yarrowia lipolytica.
  • the procedures and methods described here can be accomplished with any species of yeast, including but not limited to any species of Schizosaccharomyces species including any species of Schizosaccharomyces and preferably Schizosaccharomyces pombe.
  • yeast species such as Kluyveromyces lactis, Saccharomyces cerevisiae, Pichia pastoris, and others, can be used as a host organism.
  • Yeast cell culture techniques are well known to those having ordinary skill in the art. Exemplary methods of yeast cell culture can be found in Evans, Yeast Protocols. Springer (1996); Bill, Recombinant Protein Production in Yeast. Springer (2012); Hagan et al., Fission Yeast: A Laboratory Manual, CSH Press (2016); Konishi et al., Improvement of the transformation efficiency of Saccharomyces cerevisiae by altering carbon sources in pre-culture.
  • MSM media recipe 2 g/L sodium citrate dihydrate; 1 g/L calcium sulfate dihydrate (0.79 g/L anhydrous calcium sulfate); 42.9g/L potassium phosphate monobasic; 5.17g/L ammonium sulfate; 14.33 g/L potassium sulfate; 11.7 g/L magnesium sulfate heptahydrate; 2 mL/L PTM1trace salt solution; 0.4 ppm biotin (from 500X, 200 ppm stock); 1-2% pure glycerol or other carbon source.
  • PTM1 trace salts solution Cupric sulfate-5H2O 6.0 g; Sodium iodide 0.08 g; Manganese sulfate-H2O 3.0 g; Sodium molybdate-2H 2 O 0.2 g; Boric Acid 0.02 g; Cobalt chloride 0.5 g; Zinc chloride 20.0 g; Ferrous sulfate-7H 2 O 65.0 g; Biotin 0.2 g; Sulfuric Acid 5.0 ml; add Water to a final volume of 1 liter.
  • An illustrative composition for K An illustrative composition for K.
  • lactis defined medium is as follows: 11.83 g/L KH 2 PO 4 , 2.299 g/L K 2 HPO 4 , 20 g/L of a fermentable sugar, e.g., galactose, maltose, latotriose, sucrose, fructose or glucose and/or a sugar alcohol, for example, erythritol, hydrogenated starch hydrolysates, isomalt, lactitol, maltitol, mannitol, and xylitol, 1 g/L MgSO 4 .7H 2 O, 10 g/L (NH 4 )SO 4 , 0.33 g/L CaCl 2 .2H 2 O, 1 g/L NaCl, 1 g/L KCl, 5 mg/L CuSO 4 .5H 2 O, 30 mg/L MnSO 4 .H 2 O, 10 mg/L, ZnCl 2 , 1 mg/L KI
  • Yeast cells can be cultured in 48-well Deep-well plates, sealed after inoculation with sterile, air-permeable cover. Colonies of yeast, for example, K. lactis cultured on plates can be picked and inoculated the deep-well plates with 2.2 mL media per well, composed of DMSor. Inoculated deep-well plates can be grown for 6 days at 23.5 ⁇ C with 280 rpm shaking in a refrigerated incubator-shaker. On day 6 post-inoculation, conditioned media should be harvested by centrifugation at 4000 rpm for 10 minutes, followed by filtration using filter plate with 0.22 ⁇ M membrane, with filtered media are subject to HPLC analyses.
  • yeast transformation Yeast transformation, peptide purification, and analysis
  • An exemplary method of yeast transformation is as follows: the expression vectors carrying a CRIP ORF, a CRIP-insecticidal protein ORF, or a peptide-IA ORF, are transformed into yeast cells.
  • the expression vectors are usually linearized by specific restriction enzyme cleavage to facilitate chromosomal integration via homologous recombination.
  • the linear expression vector is then transformed into yeast cells by a chemical or electroporation method of transformation and integrated into the targeted locus of the yeast genome by homologous recombination.
  • the integration can happen at the same chromosomal locus multiple times; therefore, the genome of a transformed yeast cell can contain multiple copies of CRIP or peptide-IA expression cassettes.
  • the successfully transformed yeast cells can be identified using growth conditions that favor a selective marker engineered into the expression vector and co-integrated into yeast chromosomes with the CRIP, CRIP- insecticidal protein, or peptide-IA ORF; examples of such markers include, but are not limited to, acetamide prototrophy, zeocin resistance, geneticin resistance, nourseothricin resistance, and uracil prototrophy.
  • transgenic yeast colonies of a given transformation process will differ in their capacities to produce a CRIP ORF, a CRIP-insecticidal protein ORF, or a peptide-IA ORF,. Therefore, transgenic yeast colonies carrying the CRIP or peptide-IA transgenes should be screened for high yield strains.
  • Two effective methods for such screening each dependent on growth of small-scale cultures of the transgenic yeast to provide conditioned media samples for subsequent analysis—use reverse-phase HPLC or housefly injection procedures to analyze conditioned media samples from the positive transgenic yeast colonies.
  • the transgenic yeast cultures can be performed using 14 mL round bottom polypropylene culture tubes with 5 to 10 mL defined medium added to each tube, or in 48- well deep well culture plates with 2.2 mL defined medium added to each well.
  • the defined medium not containing crude proteinaceous extracts or by-products such as yeast extract or peptone, is used for the cultures to reduce the protein background in the conditioned media harvested for the later screening steps.
  • the cultures are performed at the optimal temperature, for example, 23.5°C for K. lactis, for about 5-6 days, until the maximum cell density is reached. CRIPs or peptide-IAs will now be produced by the transformed yeast cells and secreted out of cells to the growth medium.
  • positive yeast colonies transformed with CRIP or peptide-IA can be screened via reverse-phase HPLC (rpHPLC) screening of putative yeast colonies.
  • rpHPLC reverse-phase HPLC
  • an HPLC analytic column with bonded phase of C18 can be used.
  • Acetonitrile and water are used as mobile phase solvents, and a UV absorbance detector set at 220 nm is used for the peptide detection.
  • conditioned medium samples are loaded into the rpHPLC system and eluted with a linear gradient of mobile phase solvents.
  • the corresponding peak area of the insecticidal peptide in the HPLC chromatograph is used to quantify the CRIP or peptide-IA concentrations in the conditioned media.
  • Known amounts of pure CRIP or peptide-IA are run through the same rpHPLC column with the same HPLC protocol to confirm the retention time of the peptide and to produce a standard peptide HPLC curve for the quantification.
  • lactis cells is as follows: a CRIP ORF, a CRIP-insecticidal protein ORF, or a peptide-IA ORF, can be inserted into the expression vector, pKLAC1, and transformed into the K. lactis strain, YCT306, from New England Biolabs, Ipswich, MA, USA.
  • pKLAC1 vector is an integrative expression vector.
  • the resulting transformed colonies produced pre-propeptides comprising an ⁇ -mating factor signal peptide, a Kex2 cleavage site and mature CRIPs or peptide-IAs.
  • the ⁇ -Mating factor signal peptide guides the pre-propeptides to enter the endogenous secretion pathway, and mature CRIP or peptide-IAs are released into the growth media.
  • codon optimization for CRIP or peptide-IA expression can be performed in two rounds, for example, in the first round, based on some common features of high expression DNA sequences, multiple variants of the CRIP or peptide-IA expression ORF, expressing an ⁇ -Mating factor signal peptide, a Kex2 cleavage site and the CRIP or peptide-IA, are designed and their expression levels are evaluated in the YCT306 strain of K. lactis, resulting in an initial K. lactis expression algorithm; in a second round of optimization, additional variant CRIP or peptide-IA expression ORFs can be designed based on the initial K. lactis expression algorithm to further fine-tuned the K.
  • the resulting DNA sequence from the foregoing optimization can have an open reading frame encoding an ⁇ -MF signal peptide, a Kex2 cleavage site and a CRIP, a CRIP- insecticidal protein, or a peptide-IA, which can be cloned into the pKLAC1 vector using Hind III and Not I restriction sites, resulting in CRIP or peptide-IA expression vectors.
  • the yeast, Pichia pastoris can be transformed with a CRIP, a CRIP-insecticidal protein, or a peptide-IA, expression cassette.
  • An exemplary method for transforming P. pastoris is as follows: the vectors, pJUG ⁇ KR and pJUZ ⁇ KR, can be used to transform the CRIP or peptide-IA into P. pastoris.
  • the pJUG ⁇ KR and pJUZ ⁇ KR vectors are available from Biogrammatics, Carlsbad, California, USA. Both vectors are integrative vectors and use the uracil phosphoribosyltransferase promoter (pUPP) to enhance the heterologous transgene expression.
  • pUPP uracil phosphoribosyltransferase promoter
  • Pairs of complementary oligonucleotides, encoding the CRIP or peptide-IA are designed and synthesized for subcloning into the two yeast expression vectors. Hybridization reactions are performed by mixing the corresponding complementary oligonucleotides to a final concentration of 20 ⁇ M in 30 mM NaCl, 10 mM Tris-Cl (all final concentrations), pH 8, and then incubating at 95°C for 20 min, followed by a 9-hour incubation starting at 92°C and ending at 17°C, with 3 °C drops in temperature every 20 min.
  • the hybridization reactions will result in DNA fragments encoding CRIP or peptide-IA.
  • the two P. pastoris vectors are digested with BsaI-HF restriction enzymes, and the double stranded DNA products of the reactions are then subcloned into the linearized P. pastoris vectors using standard procedures.
  • plasmid aliquots are transfected by electroporation into the P. pastoris strain, Bg08.
  • the resulting transformed yeast selected based on resistance to Zeocin or G418 conferred by elements engineered into vectors pJUZ ⁇ KR and pJUG ⁇ KR, respectively, can be cultured and screened as described herein.
  • Peptide yield can be determined by any of the methods known to those of skill in the art (e.g., capillary gel electrophoresis (CGE), Western blot analysis, and the like). Activity assays, as described herein and known in the art, can also provide information regarding peptide yield. In some embodiments, these or any other methods known in the art can be used to evaluate peptide yield.
  • CGE capillary gel electrophoresis
  • Activity assays as described herein and known in the art, can also provide information regarding peptide yield. In some embodiments, these or any other methods known in the art can be used to evaluate peptide yield.
  • CRIP peptide yield can be measured using: HPLC; Mass spectrometry (MS) and related techniques; LC/MS/MS; reverse phase protein arrays (RPPA); immunohistochemistry; ELISA; suspension bead array, mass spectrometry; dot blot; SDS-PAGE; capillary gel electrophoresis (CGE); Western blot analysis; Bradford assay; measuring UV absorption at 260nm; Lowry assay; Smith copper/bicinchoninic assay; a secretion assay; Pierce protein assay; Biuret reaction; and the like.
  • CRIP peptide yield can be quantified and/or assessed using methods that include, without limitation: recombinant protein mass per volume of culture (e.g., gram or milligrams protein per liter culture); percent or fraction of recombinant protein insoluble precipitate obtained after cell lysis determined in (e.g., recombinant protein extracted supernatant in an amount/amount of protein in the insoluble components); percentage or fraction of active protein (e.g., an amount/analysis of the active protein for use in protein amount); total cell protein (tcp) percentage or fraction; and/or the amount of protein/cell and the dry biomass of a percentage or ratio.
  • recombinant protein mass per volume of culture e.g., gram or milligrams protein per liter culture
  • percent or fraction of recombinant protein insoluble precipitate obtained after cell lysis determined in e.g., recombinant protein extracted supernatant in an amount/amount of protein in the insoluble components
  • percentage or fraction of active protein
  • the culture cell density may be taken into account, particularly when yields between different cultures are being compared.
  • the present invention provides a method of producing a heterologous polypeptide that is at least about 5%, at least about 10%, about 15%, about 20%, about 25%, about 30%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, or greater of total cell protein (tcp).
  • tcp total cell protein
  • Total cell protein (tcp)” or “Percent total cell protein (% tcp)” is the amount of protein or polypeptide in the host cell as a percentage of aggregate cellular protein. Methods for the determination of the percent total cell protein are well known in the art.
  • HPLC can be used to quantify peptide yield.
  • CRIP or peptide-IA yield can be evaluated using an Agilent 1100 HPLC system equipped with an Onyx monolithic 4.5 x 100 mm, C18 reverse-phase analytical HPLC column and an auto-injector.
  • lactis cells are analyzed using Agilent 1100 HPLC system equipped with an Onyx monolithic 4.5 x 100 mm, C18 reverse-phase analytical HPLC column and an auto- injector by analyzing HPLC grade water and acetonitrile containing 0.1% trifluoroacetic acid, constituting the two mobile phase solvents used for the HPLC analyses; the peak areas of both the CRIP or peptide-IA are analyzed using HPLC chromatographs, and then used to calculate the peptide concentration in the conditioned media, which can be further normalized to the corresponding final cell densities (as determined by OD600 measurements) as normalized peptide yield.
  • a CRIP or a peptide-IA can be screened using a housefly injection assay.
  • the CRIP or peptide-IA can paralyze/kill houseflies when injected in measured doses through the body wall of the dorsal thorax.
  • the efficacy of the CRIP or peptide-IA can be defined by the median paralysis/lethal dose of the peptide (PD50/LD50), which causes 50% knock-down ratio or mortality of the injected houseflies respectively.
  • the pure CRIP or peptide-IA is normally used in the housefly injection assay to generate a standard dose-response curve, from which a PD 50 /LD 50 value can be determined.
  • a PD 50 /LD 50 value from the analysis of a standard dose- response curve of the pure CRIP or peptide-IA
  • quantification of the CRIP or peptide-IA produced by the transformed yeast can be achieved using a housefly injection assay performed with serial dilutions of the corresponding conditioned media.
  • An exemplary housefly injection bioassay is as follows: conditioned media is serially diluted to generate full dose-response curves from the housefly injection bioassay.
  • adult houseflies (Musca domestica) are immobilized with CO2, and 12-18 mg houseflies are selected for injection.
  • a microapplicator loaded with a 1 cc syringe and 30-gauge needle, is used to inject 0.5 ⁇ L per fly, doses of serially diluted conditioned media samples into houseflies through the body wall of the dorsal thorax.
  • the injected houseflies are placed into closed containers with moist filter paper and breathing holes on the lids, and they are examined by knock-down ratio or by mortality scoring at 24 hours post-injection. Normalized yields are calculated.
  • Peptide yield means the peptide concentration in the conditioned media in units of mg/L.
  • peptide yields are not always sufficient to accurately compare the strain production rate.
  • Individual strains may have different growth rates, hence when a culture is harvested, different cultures may vary in cell density.
  • a culture with a high cell density may produce a higher concentration of the peptide in the media, even though the peptide production rate of the strain is lower than another strain which has a higher production rate.
  • the term “normalized yield” is created by dividing the peptide yield with the cell density in the corresponding culture and this allows a better comparison of the peptide production rate between strains.
  • the cell density is represented by the light absorbance at 600 nm with a unit of “A” (Absorbance unit).
  • Screening yeast colonies that have undergone a transformation with CRIP or peptide-IA can identify the high yield yeast strains from hundreds of potential colonies. These strains can be fermented in bioreactor to achieve at least up to 4 g/L or at least up to 3 g/L or at least up to 2 g/L yield of the CRIP or peptide-IA when using optimized fermentation media and fermentation conditions described herein.
  • the higher rates of production can be anywhere from about 100 mg/L to about 100,000 mg/L; or from about 100 mg/L to about 90, 000 mg/L; or from about 100 mg/L to about 80,000 mg/L; or from about 100 mg/L to about 70,000 mg/L; or from about 100 mg/L to about 60,000 mg/L; or from about 100 mg/L to about 50,000 mg/L; or from about 100 mg/L to about 40,000 mg/L; or from about 100 mg/L to about 30,000 mg/L; or from about 100 mg/L to about 20,000 mg/L; or from about 100 mg/L to about 17,500 mg/L; or from about 100 mg/L to about 15,000 mg/L; or from about 100 mg/L to about 12,500 mg/L; or from about 100 mg/L to about 10,000 mg/L; or from about 100 mg/L to about 9,000 mg/L; or from about 100 mg/L to about 8,000 mg/L; or from about 100 mg/L to about 7,000 mg/L; or
  • Any of the foregoing methods can be used and/or tailored to produce a CRIP and/or peptide-IA (e.g., an Insecticidal Agent that lends itself to such methods, e.g., a polymer of amino acids, a peptide and/or a protein).
  • a CRIP and/or peptide-IA e.g., an Insecticidal Agent that lends itself to such methods, e.g., a polymer of amino acids, a peptide and/or a protein.
  • any of the foregoing methods can be used to produce, generate, make, express, transcribe, translate, synthesize or otherwise create, any of the CRIPs or peptide-IAs described herein, including, without limitation, ACTX peptides (e.g., U-ACTX-Hv1a, U+2-ACTX-Hv1a, rU-ACTX-Hv1a, rU- ACTX-Hv1b, r ⁇ -ACTX-Hv1c, ⁇ -ACTX-Hv1a, and/or ⁇ -ACTX-Hv1a+2); ⁇ -CNTX-Pn1a; U1-agatoxin-Ta1b; TVPs; Av2; Av3; AVPs; and/or Bt toxins (e.g., Cry toxins, Cyt toxins, or Vips).
  • ACTX peptides e.g., U-ACTX-Hv1a, U+2-ACTX-H
  • Yeast culture [00476] Yeast cell culture techniques are well known to those having ordinary skill in the art. Exemplary methods of yeast cell culture can be found in Evans, Yeast Protocols. Springer (1996); Bill, Recombinant Protein Production in Yeast.
  • yeast can be cultured in a variety of media, e.g., in some embodiments, yeast can be cultured in minimal medium; YPD medium; yeast synthetic drop-out medium; Yeast Nitrogen Base (YNB with or without amino acids); YEPD medium; ADE D medium; ADE DS" medium; LEU D medium; HIS D medium; or Mineral salts medium.
  • yeast can be cultured in minimal medium.
  • minimal medium ingredients can comprise: 2% Sugar; Phosphate Buffer, pH 6.0; Magnesium Sulfate; Calcium Chloride; Ammonium Sulfate; Sodium Chloride; Potassium Chloride; Copper Sulfate; Manganese Sulfate; Zinc Chloride; Potassium Iodide; Cobalt Chloride; Sodium Molybdate; Boric Acid; Iron Chloride; Biotin; Calcium pantothenate; Thiamine; Myo-inositol; Nicotinic Acid; and Pyridoxine.
  • yeast can be cultured in YPD medium.
  • YPD medium comprises a bacteriological peptone, yeast extract, and glucose.
  • yeast can be cultured in yeast synthetic drop-out medium, which can be used to differentiate auxotrophic mutant strains that cannot grow without a specific medium component transformed with a plasmid that allows said transformant to grow on a medium lacking the required component.
  • yeast can be cultured using Yeast Nitrogen Base (YNB with or without amino acids), which comprises nitrogen, vitamins, trace elements, and salts.
  • the medium can be YEPD medium, e.g., a medium comprising 2% D-glucose, 2% BACTO Peptone (Difco Laboratories, Detroit, MI), 1% BACTO yeast extract (Difco), 0.004% adenine, and 0.006% L-leucine; or, a variation thereof, wherein the carbon source is a sugar alcohol, e.g., glycerol or sorbitol [00483] In some embodiments, the medium can be ADE D medium, e.g., a medium comprising 0.056%-Ade-Trp-Thr powder, 0.67% yeast nitrogen base without amino acids, 2% D-glucose, and 0.5% 200 ⁇ tryptophan, threonine solution; or, a variation thereof, wherein the carbon source is a sugar alcohol, e.g., glycerol or sorbitol [00484] In some embodiments, the medium can be ADE D medium, e.g.
  • the medium can be HIS D medium, e.g., a medium comprising 0.052%-His-Trp-Thr powder, 0.67% yeast nitrogen base without amino acids, 2% D-glucose, and 0.5% 200 ⁇ tryptophan, threonine solution; or, a variation thereof, wherein the carbon source is a sugar alcohol, e.g., glycerol or sorbitol.
  • a mineral salts medium can be used.
  • Mineral salts media consists of mineral salts and a carbon source such as, e.g., glucose, sucrose, or glycerol.
  • mineral salts media examples include, e.g., M9 medium, Pseudomonas medium (ATCC 179), and Davis and Mingioli medium. See, Davis & Mingioli (1950) J. Bact.60:17- 28.
  • the mineral salts used to make mineral salts media include those selected from among, e.g., potassium phosphates, ammonium sulfate or chloride, magnesium sulfate or chloride, and trace minerals such as calcium chloride, borate, and sulfates of iron, copper, manganese, and zinc.
  • no organic nitrogen source such as peptone, tryptone, amino acids, or a yeast extract, is included in a mineral salts medium.
  • an inorganic nitrogen source is used and this may be selected from among, e.g., ammonium salts, aqueous ammonia, and gaseous ammonia.
  • a mineral salts medium will typically contain glucose or glycerol as the carbon source.
  • minimal media can also contain mineral salts and a carbon source, but can be supplemented with, e.g., low levels of amino acids, vitamins, peptones, or other ingredients, though these are added at very minimal levels.
  • Media can be prepared using the methods described in the art, e.g., in U.S. Pat. App. Pub. No. 2006/0040352, the disclosure of which is incorporated herein by reference in its entirety.
  • Kluyveromyces lactis are grown in minimal media supplemented with 2% glucose, galactose, sorbitol, or glycerol as the sole carbon source. Cultures are incubated at 30oC until mid-log phase (24-48 hours) for ⁇ -galactosidase measurements, or for 6 days at 23.5oC for heterologous protein expression.
  • yeast cells can be cultured in 48-well Deep-well plates, sealed after inoculation with sterile, air-permeable cover.
  • Colonies of yeast, for example, K. lactis cultured on plates can be picked and inoculated the deep-well plates with 2.2 mL media per well, composed of DMSor.
  • Inoculated deep-well plates can be grown for 6 days at 23.5 ⁇ C with 280 rpm shaking in a refrigerated incubator-shaker.
  • conditioned media should be harvested by centrifugation at 4000 rpm for 10 minutes, followed by filtration using filter plate with 0.22 ⁇ M membrane, with filtered media are subject to HPLC analyses.
  • yeast species such as Kluyveromyces lactis, Saccharomyces cerevisiae, Pichia pastoris, and others, can be used as a host organism, and/or the yeast to be modified using the methods described herein.
  • Temperature and pH conditions will vary depending on the stage of culture and the host cell species selected. Variables such as temperature and pH in cell culture are readily known to those having ordinary skill in the art.
  • the pH level is important in the culturing of yeast. One of skill in the art will appreciate that the culturing process includes not only the start of the yeast culture but the maintenance of the culture as well.
  • the yeast culture may be started at any pH level, however, since the media of a yeast culture tends to become more acidic (i.e., lowering the pH) over time, care must be taken to monitor the pH level during the culturing process.
  • the yeast is grown in a medium at a pH level that is dictated based on the species of yeast used, the stage of culture, and/or the temperature.
  • the pH level can fall within a range from about 2 to about 10. Those having ordinary skill in the art will recognize that the optimum pH for most microorganisms is near the neutral point (pH 7.0).
  • the pH can range from 2 to 6.5. In some embodiments, the pH can range from about 4 to about 4.5. Some fungal species (e.g., molds) can grow can grow in a pH of from about 2 to about 8.5, but favor an acid pH. See Mountney & Gould, Practical food microbiology and technology.1988. Ed.3; and Pena et al., Effects of high medium pH on growth, metabolism and transport in Saccharomyces cerevisiae. FEMS Yeast Res.2015 Mar;15(2):fou005.
  • the pH is about 5.7 to 5.9, 5.8 to 6.0, 5.9 to 6.1, 6.0 to 6.2, 6.1 to 6.3, 6.2 to 6.5, 6.4 to 6.7, 6.5 to 6.8, 6.6 to 6.9, 6.7 to 7.0, 6.8 to 7.1, 6.9 to 7.2, 7.0 to 7.3, 7.1 to 7.4, 7.2 to 7.5, 7.3 to 7.6, 7.4 to 7.7, 7.5 to 7.8, 7.6 to 7.9, 7.7 to 8.0, 7.8 to 8.1, 7.9 to 8.2, 8.0 to 8.3, 8.1 to 8.4, 8.2 to 8.5, 8.3 to 8.6, 8.4 to 8.7, or 8.5 to 8.8.
  • the pH of the medium can be at least 5.5. In other aspects, the medium can have a pH level of about 5.5. In other aspects, the medium can have a pH level of between 4 and 8. In some cases, the culture is maintained at a pH level of between 5.5 and 8. In other aspects, the medium has a pH level of between 6 and 8. In some cases, medium has a pH level that is maintained at a pH level of between 6 and 8. In some embodiments, the yeast is grown and/or maintained at a pH level of between 6.1 and 8.1. In some embodiments, the yeast is grown and/or maintained at a pH level of between 6.2 and 8.2. In some embodiments, the yeast is grown and/or maintained at a pH level of between 6.3 and 8.3.
  • the yeast is grown and/or maintained at a pH level of between 6.4 and 8.4. In some embodiments, the yeast is grown and/or maintained at a pH level of between 5.5 and 8.5. In some embodiments, the yeast is grown and/or maintained at a pH level of between 6.5 and 8.5. In some embodiments, the yeast is grown at a pH level of about 5.6, 5.7, 5.8 or 5.9. In some embodiments, the yeast is grown at a pH level of about 6. In some embodiments, the yeast is grown at a pH level of about 6.5. In some embodiments, the yeast is grown at a pH level of about 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9 or 7.0.
  • the yeast is grown at a pH level of about 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0. In some embodiments, the yeast is grown at a level of above 8. [00497] In some embodiments, the pH of the medium can range from a pH of 2 to 8.5.
  • the pH is about 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, or 8.8.
  • Exemplary methods of yeast culture can be found in U.S. Patent No.
  • fermentation may be performed at any scale.
  • the methods and techniques contemplated according to the present invention are useful for recombinant protein expression at any scale.
  • microliter-scale, milliliter scale, centiliter scale, and deciliter scale fermentation volumes may be used, and 1 Liter scale and larger fermentation volumes can be used.
  • the fermentation volume is at or above about 1 Liter.
  • the fermentation volume is about 1 liter to about 100 liters.
  • the fermentation volume is about 1 liter, about 2 liters, about 3 liters, about 4 liters, about 5 liters, about 6 liters, about 7 liters, about 8 liters, about 9 liters, or about 10 liters.
  • the fermentation volume is about 1 liter to about 5 liters, about 1 liter to about 10 liters, about 1 liter to about 25 liters, about 1 liter to about 50 liters, about 1 liter to about 75 liters, about 10 liters to about 25 liters, about 25 liters to about 50 liters, or about 50 liters to about 100 liters
  • the fermentation volume is at or above 5 Liters, 10 Liters, 15 Liters, 20 Liters, 25 Liters, 50 Liters, 75 Liters, 100 Liters, 200 Liters, 500 Liters, 1,000 Liters, 2,000 Liters, 5,000 Liters, 10,000 Liters, or 50,000 Liters.
  • the fermentation medium can be a nutrient solution used for growing and or maintaining cells.
  • this solution ordinarily provides at least one component from one or more of the following categories: (1) an energy source, usually in the form of a carbon source, e.g., glucose; (2) all essential amino acids, and usually the basic set of twenty amino acids; (3) vitamins and/or other organic compounds required at low concentrations; (4) free fatty acids or lipids, for example linoleic acid; and (5) trace elements, where trace elements are defined as inorganic compounds or naturally occurring elements that are typically required at very low concentrations, usually in the micromolar range.
  • the fermentation medium can be the same as the cell culture medium or any other media described herein. In some embodiments, the fermentation medium can be different from the cell culture medium. In some embodiments, the fermentation medium can be modified in order to accommodate the large-scale production of proteins.
  • the fermentation medium can be supplemented electively with one or more components from any of the following categories: (1) hormones and other growth factors such as, serum, insulin, transferrin, and the like; (2) salts, for example, magnesium, calcium, and phosphate; (3) buffers, such as HEPES; (4) nucleosides and bases such as, adenosine, thymidine, etc.; (5) protein and tissue hydrolysates, for example peptone or peptone mixtures which can be obtained from purified gelatin, plant material, or animal byproducts; (6) antibiotics, such as gentamycin; and (7) cell protective agents, for example pluronic polyol.
  • hormones and other growth factors such as, serum, insulin, transferrin, and the like
  • salts for example, magnesium, calcium, and phosphate
  • buffers such as HEPES
  • nucleosides and bases such as, adenosine, thymidine, etc.
  • protein and tissue hydrolysates for example peptone or
  • the pH of the fermentation medium can be maintained using pH buffers and methods known to those of skill in the art. Control of pH during fermentation can also can be achieved using aqueous ammonia.
  • the pH of the fermentation medium will be selected based on the preferred pH of the organism used. Thus, in some embodiments, and depending on the host cell and temperature, the pH can range from about to 1 to about 10.
  • the pH of the fermentation medium can range from a pH of 2 to 8.5.
  • the pH is about 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, or 8.8.
  • the pH is about 5.7 to 5.9, 5.8 to 6.0, 5.9 to 6.1, 6.0 to 6.2, 6.1 to 6.3, 6.2 to 6.5, 6.4 to 6.7, 6.5 to 6.8, 6.6 to 6.9, 6.7 to 7.0, 6.8 to 7.1, 6.9 to 7.2, 7.0 to 7.3, 7.1 to 7.4, 7.2 to 7.5, 7.3 to 7.6, 7.4 to 7.7, 7.5 to 7.8, 7.6 to 7.9, 7.7 to 8.0, 7.8 to 8.1, 7.9 to 8.2, 8.0 to 8.3, 8.1 to 8.4, 8.2 to 8.5, 8.3 to 8.6, 8.4 to 8.7, or 8.5 to 8.8 [00509] In some embodiments, e.g., where Escherichia coli (E.
  • the optimal pH range is between 6.5 and 7.5, depending on the temperature.
  • the pH can range from about 4.0 to 8.0.
  • neutral pH i.e., a pH of about 7.0 can be used.
  • the fermentation medium can be supplemented with a buffer or other chemical in order to avoid changes to the pH.
  • the addition of Ca(OH) 2 , CaCO 3 , NaOH, or NH 4 OH can be added to the fermentation medium to neutralize the production of acidic compounds that occur, e.g., in some yeast species during industrial processes.
  • Temperature is another important consideration in the fermentation process; and, like pH considerations, temperature will depend on the type of host cell selected.
  • the fermentation temperature is maintained at about 4°C. to about 42°C.
  • the fermentation temperature is about 4°C, about 5°C, about 6°C, about 7°C, about 8°C, about 9°C, about 10°C, about 11°C, about 12°C, about 13°C, about 14°C, about 15°C, about 16°C, about 17°C, about 18°C, about 19°C, about 20°C, about 21°C, about 22°C, about 23°C, about 24°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 31°C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, about 40°C, about 41°C, or about 42°C.
  • the fermentation temperature is maintained at about 25°C to about 27°C, about 25°C to about 28°C, about 25°C to about 29°C, about 25°C to about 30°C, about 25°C to about 31°C, about 25°C to about 32°C, about 25°C to about 33°C, about 26°C to about 28°C, about 26°C to about 29°C, about 26°C to about 30°C, about 26°C to about 31°C, about 26°C to about 32°C, about 27°C to about 29°C, about 27°C to about 30°C, about 27°C to about 31°C, about 27°C to about 32°C, about 26°C to about 33°C, about 28°C to about 30°C, about 28°C to about 31°C, about 28°C to about 32°C, about 29°C to about 31°C, about 29°C to about 32°C, about 29°C to about 33°C, about 30°C to about 32°C,
  • microorganisms for up-scaled production of a CRIP, a CRIP-insecticidal protein, or a peptide-IA include any microorganism listed herein.
  • non-limiting examples of microorganisms include strains of the genus Saccharomyces spp. (including, but not limited to, S. cerevisiae (baker's yeast), S. distaticus, S. uvarum), the genus Kluyveromyces, (including, but not limited to, K. marxianus, K.
  • genus Candida including, but not limited to, C. pseudotropicalis, and C. brassicae
  • Pichia stipitis a relative of Candida shehatae
  • the genus Clavispora including, but not limited to, C. lusitaniae and C. opuntiae
  • the genus Pachysolen including, but not limited to, P. tannophilus
  • the genus Bretannomyces including, but not limited to, e.g., B. clausenii.
  • Other suitable microorganisms include, for example, Zymomonas mobilis, Clostridium spp. (including, but not limited to, C. thermocellum; C. saccharobutylacetonicum, C.
  • Fermentation medium may be selected depending on the host cell and/or needs of the end-user. Any necessary supplements besides, e.g., carbon, may also be included at appropriate concentrations introduced alone or as a mixture with another supplement or medium such as a complex nitrogen source.
  • Yeast Fermentation Fermentation methods using yeast are well known to those having ordinary skill in the art.
  • batch fermentation can be used according to the methods provided herein; in other embodiments, continuous fermentation procedures can be used.
  • the batch method of fermentation can be used to produce CRIPs, CRIP-insecticidal proteins, or peptide-IAs of the present invention.
  • the batch method of fermentation refers to a type of fermentation that is performed with a closed system, wherein the composition of the medium is determined at the beginning of the fermentation and is not subject to artificial alterations during the fermentation (i.e., the medium is inoculated with one or more yeast cells at the start of fermentation, and fermentation is allowed to proceed, uninterrupted by the user).
  • yeast cells typically pass through a static lag phase to a high growth log phase, and, finally, to a stationary phase, in which the growth rate is diminished or stopped. If untreated, yeast cells in the stationary phase will eventually die.
  • yeast cells in log phase generally are responsible for the bulk of synthesis of end product.
  • fed-batch fermentation can be used to produce CRIPs, CRIP-insecticidal proteins, or peptide-IAs of the present invention.
  • fed-batch fermentation is similar to typical batch method (described above), however, the substrate in the fed-batch method is added in increments as the fermentation progresses.
  • Fed-batch fermentation is useful when catabolite repression may inhibit yeast cell metabolism, and when it is desirable to have limited amounts of substrate in the medium.
  • the measurement of the substrate concentration in a fed-batch system is estimated on the basis of the changes of measurable factors reflecting metabolism, such as pH, dissolved oxygen, the partial pressure of waste gases (e.g., CO2), and the like.
  • the fed-batch fermentation procedure can be used to produce CRIPs, CRIP-insecticidal proteins, or peptide-IAs as follows: culturing a production organism (e.g., a modified yeast cell) in a 10 L bioreactor sparged with an N2/CO2 mixture, using 5 L broth containing 5 g/L potassium phosphate, 2.5 g/L ammonium chloride, 0.5 g/L magnesium sulfate, and 30 g/L corn steep liquor, and an initial first and second carbon source concentration of 20 g/L. As the modified yeast cells grow and utilize the carbon sources, additional 70% carbon source mixture is then fed into the bioreactor at a rate approximately balancing carbon source consumption.
  • a production organism e.g., a modified yeast cell
  • the temperature of the bioreactor is generally maintained at 30° C. Growth continues for approximately 24 hours or more, and the heterologous peptides reach a desired concentration, e.g., with the cell density being between about 5 and 10 g/L.
  • the fermenter contents can be passed through a cell separation unit such as a centrifuge to remove cells and cell debris, and the fermentation broth can be transferred to a product separations unit. Isolation of the heterologous peptides can take place by standard separations procedures well known in the art.
  • continuous fermentation can be used to produce CRIPs, CRIP-insecticidal proteins, or peptide-IAs of the present invention.
  • continuous fermentation refers to fermentation with an open system, wherein a fermentation medium is added continuously to a bioreactor, and an approximately equal amount of conditioned medium is removed simultaneously for processing.
  • Continuous fermentation generally maintains the cultures at a high density, in which yeast cells are primarily in log phase growth.
  • continuous fermentation methods are performed to maintain steady state growth conditions, and yeast cell loss, due to medium withdrawal, should be balanced against the cell growth rate in the fermentation.
  • the continuous fermentation method can be used to produce CRIPs, CRIP-insecticidal proteins, or peptide-IAs as follows: a modified yeast strain can be cultured using a bioreactor apparatus and a medium composition, albeit where the initial first and second carbon source is about, e.g., 30-50 g/L. When the carbon source is exhausted, feed medium of the same composition is supplied continuously at a rate of between about 0.5 L/hr and 1 L/hr, and liquid is withdrawn at the same rate. The heterologous peptide concentration in the bioreactor generally remains constant along with the cell density.
  • the bioreactor when producing CRIPs, CRIP-insecticidal proteins, or peptide-IAs, can be operated continuously, for example, for about one month, with samples taken every day or as needed to assure consistency of the target chemical compound concentration. In continuous mode, fermenter contents are constantly removed as new feed medium is supplied. The exit stream, containing cells, medium, and heterologous peptides, can then be subjected to a continuous product separations procedure, with or without removing cells and cell debris, and can be performed by continuous separations methods well known in the art to separate organic products from peptides of interest.
  • a yeast cell operable to express a CRIP, a CRIP- insecticidal protein, or a peptide-IA can be grown, e.g., using a fed batch process in aerobic bioreactor. Briefly, reactors are filled to about 20% to about 70% capacity with medium comprising a carbon source and other reagents. Temperature and pH is maintained using one or more chemicals as described herein. Oxygen level is maintained by sparging air intermittently in concert with agitation.
  • the present invention provides a method of using a fed batch process in aerobic bioreactor, wherein the reactor is filled to about 20%; 21%; 22%; 23%; 24%; 25%; 26%; 27%; 28%; 29%; 30%; 31%; 32%; 33%; 34%; 35%; 36%; 37%; 38%; 39%; 40%; 41%; 42%; 43%; 44%; 45%; 46%; 47%; 48%; 49%; 50%; 51%; 52%; 53%; 54%; 55%; 56%; 57%; 58%; 59%; 60%; 61%; 62%; 63%; 64%; 65%; 66%; 67%; 68%; 69%; or 70% capacity.
  • the present invention provides a fed batch fermentation method using an aerobic bioreactor to produce CRIPs, CRIP-insecticidal proteins, or peptide- IAs, wherein the medium is a rich culture medium.
  • the carbon source can be glucose, sorbitol, or lactose.
  • the amount of glucose can be about 2 g/L; 3 g/L; 4 g/L; 5 g/L; 6 g/L; 7 g/L; 8 g/L; 9 g/L; 10 g/L; 11 g/L; 12 g/L; 13 g/L; 14 g/L; 15 g/L; 16 g/L; 17 g/L; 18 g/L; 19 g/L; 20 g/L; 21 g/L; 22 g/L; 23 g/L; 24 g/L; 25 g/L; 26 g/L; 27 g/L; 28 g/L; 29 g/L; or 30 g/L of the medium.
  • the amount of sorbitol can be about 2 g/L; 3 g/L; 4 g/L; 5 g/L; 6 g/L; 7 g/L; 8 g/L; 9 g/L; 10 g/L; 11 g/L; 12 g/L; 13 g/L; 14 g/L; 15 g/L; 16 g/L; 17 g/L; 18 g/L; 19 g/L; 20 g/L; 21 g/L; 22 g/L; 23 g/L; 24 g/L; 25 g/L; 26 g/L; 27 g/L; 28 g/L; 29 g/L; or 30 g/L of the medium.
  • the amount of lactose can be about 2 g/L; 3 g/L; 4 g/L; 5 g/L; 6 g/L; 7 g/L; 8 g/L; 9 g/L; 10 g/L; 11 g/L; 12 g/L; 13 g/L; 14 g/L; 15 g/L; 16 g/L; 17 g/L; 18 g/L; 19 g/L; 20 g/L; 21 g/L; 22 g/L; 23 g/L; 24 g/L; 25 g/L; 26 g/L; 27 g/L; 28 g/L; 29 g/L; or 30 g/L of the medium.
  • the present invention provides a fed batch fermentation method using an aerobic bioreactor, wherein the medium is supplemented with one or more of phosphoric acid, calcium sulfate, potassium sulfate, magnesium sulfate heptahydrate, potassium hydroxide, and/or corn steep liquor.
  • the medium can be supplemented with phosphoric acid in an amount of about 2 g/L; 3 g/L; 4 g/L; 5 g/L; 6 g/L; 7 g/L; 8 g/L; 9 g/L; 10 g/L; 11 g/L; 12 g/L; 13 g/L; 14 g/L; 15 g/L; 16 g/L; 17 g/L; 18 g/L; 19 g/L; 20 g/L; 21 g/L; 22 g/L; 23 g/L; 24 g/L; 25 g/L; 26 g/L; 27 g/L; 28 g/L; 29 g/L; or 30 g/L to the medium.
  • the medium can be supplemented with calcium sulfate in an amount of about 0.05 g/L; 0.15 g/L; 0.25 g/L; 0.35 g/L; 0.45 g/L; 0.55 g/L; 0.65 g/L; 0.75 g/L; 0.85 g/L; 0.95 g/L; 1.05 g/L; 1.15 g/L; 1.25 g/L; 1.35 g/L; 1.45 g/L; 1.55 g/L; 1.65 g/L; 1.75 g/L; 1.85 g/L; 1.95 g/L; 2.05 g/L; 2.15 g/L; 2.25 g/L; 2.35 g/L; 2.45 g/L; 2.55 g/L; 2.65 g/L; 2.75 g/L; 2.85 g/L; or 2.95 g/L to the medium.
  • the medium can be supplemented with potassium sulfate in an amount of about 2 g/L; 2.5 g/L; 3 g/L; 3.5 g/L; 4 g/L; 4.5 g/L; 5 g/L; 5.5 g/L; 6 g/L; 6.5 g/L; 7 g/L; 7.5 g/L; 8 g/L; 8.5 g/L; 9 g/L; 9.5 g/L; 10 g/L; 10.5 g/L; 11 g/L; 11.5 g/L; 12 g/L; 12.5 g/L; 13 g/L; 13.5 g/L; 14 g/L; 14.5 g/L; 15 g/L; 15.5 g/L; 16 g/L; 16.5 g/L; 17 g/L; 17.5 g/L; 18 g/L; 18.5 g/L; 19 g/L;
  • the medium can be supplemented with magnesium sulfate heptahydrate in an amount of about 0.25 g/L; 0.5 g/L; 0.75 g/L; 1 g/L; 1.25 g/L; 1.5 g/L; 1.75 g/L; 2 g/L; 2.25 g/L; 2.5 g/L; 2.75 g/L; 3 g/L; 3.25 g/L; 3.5 g/L; 3.75 g/L; 4 g/L; 4.25 g/L; 4.5 g/L; 4.75 g/L; 5 g/L; 5.25 g/L; 5.5 g/L; 5.75 g/L; 6 g/L; 6.25 g/L; 6.5 g/L; 6.75 g/L; 7 g/L; 7.25 g/L; 7.5 g/L; 7.75 g/L; 8 g/L; 8.25 g/L;
  • the medium can be supplemented with potassium hydroxide in an amount of about 0.25 g/L; 0.5 g/L; 0.75 g/L; 1 g/L; 1.25 g/L; 1.5 g/L; 1.75 g/L; 2 g/L; 2.25 g/L; 2.5 g/L; 2.75 g/L; 3 g/L; 3.25 g/L; 3.5 g/L; 3.75 g/L; 4 g/L; 4.25 g/L; 4.5 g/L; 4.75 g/L; 5 g/L; 5.25 g/L; 5.5 g/L; 5.75 g/L; 6 g/L; 6.25 g/L; 6.5 g/L; 6.75 g/L; or 7 g/L to the medium.
  • the medium can be supplemented with corn steep liquor in an amount of about 5 g/L; 6 g/L; 7 g/L; 8 g/L; 9 g/L; 10 g/L; 11 g/L; 12 g/L; 13 g/L; 14 g/L; 15 g/L; 16 g/L; 17 g/L; 18 g/L; 19 g/L; 20 g/L; 21 g/L; 22 g/L; 23 g/L; 24 g/L; 25 g/L; 26 g/L; 27 g/L; 28 g/L; 29 g/L; 30 g/L; 31 g/L; 32 g/L; 33 g/L; 34 g/L; 35 g/L; 36 g/L; 37 g/L; 38 g/L; 39 g/L; 40 g/L; 41 g/L; 42 g/L; 43 g/L; 40 g/
  • the temperature of the reactor can be maintained between about 15°C and about 45°C.
  • the reactor can have a temperature of about 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, or 40°C.
  • the pH can have a level of about 3 to about 6.
  • the pH can be 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or 6.0.
  • the pH can be maintained at a constant level via the addition of one or more chemicals.
  • ammonium hydroxide can be added to maintain pH.
  • ammonium hydroxide can be added to a level of ammonium hydroxide in the medium that is about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%, of ammonium hydroxide [00544]
  • oxygen levels can be maintained by sparging.
  • dissolved oxygen can be maintained at a constant level by sparging air between 0.5-1.5 volume/volume/min and by increasing agitation to maintain a set point of 10-30%.
  • inoculation of the reactor can be accomplished based on an overnight seed culture comprising from about 2.5 g/L to about 50 g/L of a carbon source, e.g., glucose, sorbitol, or lactose.
  • the overnight seed culture can comprise corn steep liquor, e.g., from about 2.5 g/L to about 50 g/L of corn steep liquor.
  • the inoculation percentage can range from about 5-20% of initial fill volume.
  • the reactor can be fed with from about a 50% to about an 80% solution of the selected carbon source up until the reactor is filled and/or the desired supernatant peptide concentration is achieved.
  • the time required to fill the reactor can range from about 86 hours to about 160 hours. In some embodiments, the quantity required to reach the desired peptide concentration can range from about 0.8 g/L to about 1.2 g/L.
  • the contents can be passed through a cell separation unit and optionally concentrated, depending on intended use of the material. [00547] Additional recipes for yeast fermentation media are provided herein.
  • MSM media recipe 2 g/L sodium citrate dihydrate; 1 g/L calcium sulfate dihydrate (0.79 g/L anhydrous calcium sulfate); 42.9g/L potassium phosphate monobasic; 5.17g/L ammonium sulfate; 14.33 g/L potassium sulfate; 11.7 g/L magnesium sulfate heptahydrate; 2 mL/L PTM1trace salt solution; 0.4 ppm biotin (from 500X, 200 ppm stock); 1-2% pure glycerol or other carbon source.
  • PTM1 trace salts solution Cupric sulfate-5H2O 6.0 g; Sodium iodide 0.08 g; Manganese sulfate-H2O 3.0 g; Sodium molybdate-2H 2 O 0.2 g; Boric Acid 0.02 g; Cobalt chloride 0.5 g; Zinc chloride 20.0 g; Ferrous sulfate-7H 2 O 65.0 g; Biotin 0.2 g; Sulfuric Acid 5.0 ml; add Water to a final volume of 1 liter.
  • An illustrative composition for K An illustrative composition for K.
  • lactis defined medium is as follows: 11.83 g/L KH 2 PO 4 , 2.299 g/L K 2 HPO 4 , 20 g/L of a fermentable sugar, e.g., galactose, maltose, latotriose, sucrose, fructose or glucose and/or a sugar alcohol, for example, erythritol, hydrogenated starch hydrolysates, isomalt, lactitol, maltitol, mannitol, and xylitol, 1 g/L MgSO 4 .7H 2 O, 10 g/L (NH 4 )SO 4 , 0.33 g/L CaCl 2 .2H 2 O, 1 g/L NaCl, 1 g/L KCl, 5 mg/L CuSO 4 .5H 2 O, 30 mg/L MnSO 4 .H 2 O, 10 mg/L, ZnCl 2 , 1 mg/L KI
  • Peptide degradation Proteins, polypeptides, and peptides degrade in both biological samples and in solution (e.g., cell culture and/or during fermentation). Methods of detecting peptide degradation (e.g., degradation of a CRIP, a CRIP-insecticidal protein, or a peptide-IA) are well known in the art. Any of the well-known methods of detecting peptide degradation (e.g., during fermentation) may be employed here.
  • peptide degradation can be detected using isotope labeling techniques; liquid chromatography/mass spectrometry (LC/MS); HPLC; radioactive amino acid incorporation and subsequent detection, e.g., via scintillation counting; the use of a reporter protein, e.g., a protein that can be detected (e.g., by fluorescence, spectroscopy, luminometry, etc.); fluorescent intensity of one or more bioluminescent proteins and/or fluorescent proteins and/or fusions thereof; pulse-chase analysis (e.g., pulse-labeling a cell with radioactive amino acids and following the decay of the labeled protein while chasing with unlabeled precursor, and arresting protein synthesis and measuring the decay of total protein levels with time); cycloheximide-chase assays; [00552] In some embodiments, an assay can be used to detect peptide degradation, wherein a sample is contacted with a non-fluorescent compound
  • non-fluorescent compounds that can be utilized as fluorescent tags for free amines according to the present disclosure are 3-(4-carboxybenzoyl) quinoline-2-carboxaldehyde (CBQCA), fluorescamine, and o-phthaldialdehyde.
  • CBQCA 3-(4-carboxybenzoyl) quinoline-2-carboxaldehyde
  • fluorescamine fluorescamine
  • o-phthaldialdehyde o-phthaldialdehyde.
  • the method to determine the readout signal from the reporter protein depends from the nature of the reporter protein.
  • the readout signal corresponds to the intensity of the fluorescent signal.
  • the readout signal may be measured using spectroscopy-, fluorometry-, photometry-, and/or luminometry-based methods and detection systems, for example. Such methods and detection systems are well known in the art.
  • peptide degradation can be detected in a sample using immunoassays that employ a detectable antibody.
  • immunoassays include, for example, agglutination assays, ELISA, Pandex microfluorimetric assay, flow cytometry, serum diagnostic assays, and immunohistochemical staining procedures, all of which are well- known in the art.
  • the levels (e.g., of fluorescence) in one sample can be compared to a standard.
  • An antibody can be made detectable by various means well known in the art.
  • a detectable marker can be directly or indirectly attached to the antibody.
  • useful markers include, for example, radionucleotides, enzymes, fluorogens, chromogens and chemiluminescent labels.
  • Exemplary methods of detecting peptide degradation is provided in U.S. Patent Nos.5,766,927; 7,504,253; 9,201,073; 9,429,566; United States Patent Application 20120028286; Eldeeb et al., A molecular toolbox for studying protein degradation in mammalian cells. J Neurochem.2019 Nov;151(4):520-533; and Buchanan et al., Cycloheximide Chase Analysis of Protein Degradation in Saccharomyces cerevisiae.
  • the CRIPs, CRIP-insecticidal proteins, and peptide-IAs described herein, and/or an insecticidal protein comprising at least one CRIP or peptide-IA as described herein, can be incorporated into plants, plant tissues, plant cells, plant seeds, and/or plant parts thereof, for either the stable, or transient expression of a CRIP, a CRIP-insecticidal protein, or a peptide-IA, and/or a polynucleotide sequence encoding the same.
  • the CRIP or peptide-IA can be incorporated into a plant using recombinant techniques known in the art.
  • the CRIP or peptide- IA or insecticidal protein comprising at least one CRIP or peptide-IA may be in the form of an insecticidal protein which may comprise one or more CRIP or peptide-IA monomers.
  • the insecticidal protein comprising at least one CRIP or peptide-IA may also comprise one or more non-CRIP or non-IA polypeptides or proteins, e.g. an endoplasmic reticulum signal peptide operably linked to one or more CRIPs or peptide-IAs.
  • CRIP or “peptide-IA” also encompasses an insecticidal protein comprising one or more CRIPs or peptide-IAs in addition to one or more non-CRIP or non-IA peptides, polypeptides or proteins
  • CRIP polynucleotide or “IA polynucleotide” is similarly also used to encompass a polynucleotide or group of polynucleotides operable to express and/or encode an insecticidal protein comprising one or more CRIPs or peptide-IAs in addition to one or more non-CRIP or non-IA polypeptides or proteins.
  • the goal of incorporating a CRIP, a CRIP-insecticidal protein, or a peptide- IA, into plants is to deliver insecticidal proteins to the pest via the insect’s consumption of the transgenic CRIP, CRIP- insecticidal protein, or peptide-IA expressed in a plant tissue consumed by the insect.
  • the consumed CRIP, CRIP-insecticidal protein, or peptide-IA may have the ability to inhibit the growth, impair the movement, or even kill an insect.
  • transgenic plants expressing a CRIP, a CRIP-insecticidal protein, or a peptide-IA polynucleotide and/or a CRIP, a CRIP-insecticidal protein, or a peptide-IA polypeptide may express said CRIP, CRIP-insecticidal protein, or peptide-IA polynucleotide/polypeptide in a variety of plant tissues, including but not limited to, the epidermis (e.g., mesophyll); periderm; phloem; xylem; parenchyma; collenchyma; sclerenchyma; and primary and secondary meristematic tissues.
  • the epidermis e.g., mesophyll
  • periderm periderm
  • phloem xylem
  • parenchyma collenchyma
  • sclerenchyma and primary
  • a polynucleotide sequence encoding a CRIP, a CRIP-insecticidal protein, or a peptide-IA can be operably linked to a regulatory region containing a phosphoenolpyruvate carboxylase promoter, resulting in the expression of a CRIP, a CRIP-insecticidal protein, or a peptide-IA in a plant’s mesophyll tissue.
  • Transgenic plants expressing a CRIP, a CRIP-insecticidal protein, or a peptide-IA and/or a polynucleotide operable to express CRIP/peptide-IA can be generated by any one of the various methods and protocols well known to those having ordinary skill in the art; such methods of the invention do not require that a particular method for introducing a nucleotide construct to a plant be used, only that the nucleotide construct gains access to the interior of at least one cell of the plant.
  • Methods for introducing nucleotide constructs into plants are known in the art including, but not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods.
  • Transgenic plants or “transformed plants” or “stably transformed” plants or cells or tissues refers to plants that have incorporated or integrated exogenous nucleic acid sequences or DNA fragments into the plant cell. These nucleic acid sequences include those that are exogenous, or not present in the untransformed plant cell, as well as those that may be endogenous, or present in the untransformed plant cell. “Heterologous” generally refers to the nucleic acid sequences that are not endogenous to the cell or part of the native genome in which they are present, and have been added to the cell by infection, transfection, microinjection, electroporation, microprojection, or the like. [00566] Transformation of plant cells can be accomplished by one of several techniques known in the art.
  • a construct that expresses an exogenous or heterologous peptide or polypeptide of interest would contain a promoter to drive transcription of the gene, as well as a 3’ untranslated region to allow transcription termination and polyadenylation.
  • a gene can be engineered such that the resulting peptide is secreted, or otherwise targeted within the plant cell to a specific region and/or organelle.
  • the gene can be engineered to contain a signal peptide to facilitate transfer of the peptide to the endoplasmic reticulum.
  • a plant expression cassette can be inserted into a plant transformation vector.
  • This plant transformation vector may be comprised of one or more DNA vectors needed for achieving plant transformation.
  • Binary vectors as well as vectors with helper plasmids are most often used for Agrobacterium-mediated transformation, where the size and complexity of DNA segments needed to achieve efficient transformation is quite large, and it is advantageous to separate functions onto separate DNA molecules.
  • Binary vectors typically contain a plasmid vector that contains the cis-acting sequences required for T-DNA transfer (such as left border and right border), a selectable marker that is engineered to be capable of expression in a plant cell, and a “gene of interest” (a gene engineered to be capable of expression in a plant cell for which generation of transgenic plants is desired). Also present on this plasmid vector are sequences required for bacterial replication. The cis-acting sequences are arranged in a fashion to allow efficient transfer into plant cells and expression therein. For example, the selectable marker gene and the CRIP/peptide-IA are located between the left and right borders.
  • a second plasmid vector contains the trans-acting factors that mediate T-DNA transfer from Agrobacterium to plant cells.
  • This plasmid often contains the virulence functions (Vir genes) that allow infection of plant cells by Agrobacterium, and transfer of DNA by cleavage at border sequences and vir-mediated DNA transfer, as is understood in the art (Hellens and Mullineaux (2000) Trends in Plant Science 5:446-451).
  • Several types of Agrobacterium strains e.g. LBA4404, GV3101, EHA101, EHA105, etc.
  • the second plasmid vector is not necessary for transforming the plants by other methods such as microprojection, microinjection, electroporation, polyethylene glycol, etc.
  • plant transformation methods involve transferring heterologous DNA into target plant cells (e.g. immature or mature embryos, suspension cultures, undifferentiated callus, protoplasts, etc.), followed by applying a maximum threshold level of appropriate selection (depending on the selectable marker gene) to recover the transformed plant cells from a group of untransformed cell mass.
  • target plant cells e.g. immature or mature embryos, suspension cultures, undifferentiated callus, protoplasts, etc.
  • a maximum threshold level of appropriate selection depending on the selectable marker gene
  • Transformation protocols as well as protocols for introducing nucleotide sequences into plants may vary depending on the type of plant or plant cell, i.e., monocot or dicot, targeted for transformation.
  • Generation of transgenic plants may be performed by one of several methods, including, but not limited to, microinjection, electroporation, direct gene transfer, introduction of heterologous DNA by Agrobacterium into plant cells (Agrobacterium-mediated transformation), bombardment of plant cells with heterologous foreign DNA adhered to particles, ballistic particle acceleration, aerosol beam transformation, Lec1 transformation, and various other non-particle direct-mediated methods to transfer DNA.
  • Chloroplasts can also be readily transformed, and methods concerning the transformation of chloroplasts are known in the art. See, for example, Svab et al. (1990) Proc. Natl. Acad. Sci. USA 87:8526-8530; Svab and Maliga (1993) Proc. Natl. Acad. Sci.
  • the method of chloroplast transformation relies on particle gun delivery of DNA containing a selectable marker and targeting of the DNA to the plastid genome through homologous recombination. Additionally, plastid transformation can be accomplished by transactivation of a silent plastid-borne transgene by tissue-preferred expression of a nuclear-encoded and plastid-directed RNA polymerase. Such a system has been reported in McBride et al. (1994) Proc. Natl. Acad. Sci. USA 91:7301- 7305.
  • heterologous foreign DNA Following integration of heterologous foreign DNA into plant cells, one having ordinary skill may then apply a maximum threshold level of appropriate selection chemical/reagent (e.g., an antibiotic) in the medium to kill the untransformed cells, and separate and grow the putatively transformed cells that survive from this selection treatment by transferring said surviving cells regularly to a fresh medium.
  • appropriate selection chemical/reagent e.g., an antibiotic
  • an artisan identifies and proliferates the cells that are transformed with the plasmid vector.
  • Molecular and biochemical methods can then be used to confirm the presence of the integrated heterologous gene of interest into the genome of the transgenic plant.
  • the cells that have been transformed may be grown into plants in accordance with conventional methods known to those having ordinary skill in the art.
  • transformed seed also referred to as “transgenic seed” having a nucleotide construct of the invention, for example, an expression cassette of the invention, stably incorporated into their genome.
  • the present disclosure provides an insecticidal protein comprising at least one CRIP/peptide-IA, that act as substrates for insect proteinases, proteases and peptidases (collectively referred to herein as “proteases”) as described above.
  • transgenic plants or parts thereof that may be receptive to the expression of CRIPs or peptide-IAs and/or compositions comprising a CRIP, a CRIP- insecticidal protein, or a peptide-IA, as described herein, can include: alfalfa, banana, barley, bean, broccoli, cabbage, canola, carrot, cassava, castor, cauliflower, celery, chickpea, Chinese cabbage, citrus, coconut, coffee, corn, clover, cotton, a cucurbit, cucumber, Douglas fir, eggplant, eucalyptus, flax, garlic, grape, hops, leek, lettuce, Loblolly pine, millets, melons, nut, oat, olive, onion, ornamental, palm, pasture grass, pea, peanut, pepper, pigeonpea, pine, potato, poplar, pumpkin, Radiata pine, radish, rapeseed, rice, rootstocks, rye, sa
  • the transgenic plant may be grown from cells that were initially transformed with the DNA constructs described herein.
  • the transgenic plant may express the encoded CRIP/peptide-IA compositions in a specific tissue, or plant part, for example, a leaf, a stem a flower, a sepal, a fruit, a root, or a seed or combinations thereof.
  • Any of the foregoing plant incorporation methods/techniques can be used to produce a CRIP and/or peptide-IA (e.g., an Insecticidal Agent that lends itself to such methods, e.g., a polymer of amino acids, a peptide or a protein).
  • any of the foregoing methods can be used to produce, generate, make, express, transcribe, translate, synthesize or otherwise create, any of the CRIPs or peptide-IAs described herein, including, without limitation, ACTX peptides (e.g., U-ACTX-Hv1a, U+2-ACTX-Hv1a, rU-ACTX- Hv1a, rU-ACTX-Hv1b, r ⁇ -ACTX-Hv1c, ⁇ -ACTX-Hv1a, and/or ⁇ -ACTX-Hv1a+2); ⁇ - CNTX-Pn1a; U1-agatoxin-Ta1b; TVPs; Av2; Av3; AVPs; and/or Bt toxins (e.g., Cry toxins, Cyt toxins, or Vips).
  • ACTX peptides e.g., U-ACTX-Hv1a, U+2-ACTX-Hv
  • the insecticidal protein comprising at least one CRIP can be operably linked to a cleavable peptide. In other embodiments, the insecticidal protein comprising at least one CRIP (or peptide-IA) can be operably linked to a non-cleavable peptide.
  • the insecticidal protein comprising at least one CRIP/peptide-IA can have two or more cleavable peptides, wherein the insecticidal protein comprises an insect cleavable linker (L), the insect cleavable linker being fused in frame with a construct comprising (CRIP-L) n , wherein “n” is an integer ranging from 1 to 200, or from 1 to 100, or from 1 to 10.
  • the insecticidal protein comprising at least one CRIP, and described herein, comprises an endoplasmic reticulum signal peptide (ERSP) operably linked with a CRIP, which is operably linked with an insect cleavable linker (L) and/or a repeat construct (L-CRIP) n or (CRIP-L) n , wherein n is an integer ranging from 1 to 200, or from 1 to 100, or from 1 to 10.
  • an exemplary insecticidal protein can include a protein construct comprising: (ERSP)-(CRIP-L) n ; (ERSP)-(L)-(CRIP-L) n ; (ERSP)-(L- CRIP) n ; (ERSP)-(L-CRIP) n -(L); wherein n is an integer ranging from 1 to 200 or from 1 to 100, or from 1 to 10.
  • a CRIP is the aforementioned U1-agatoxin-Ta1b Variant Polypeptides
  • L is a non-cleavable or cleavable peptide
  • n is an integer ranging from 1 to 200, preferably an integer ranging from 1 to 100, and more preferably an integer ranging from 1 to 10.
  • the insecticidal protein may contain CRIP peptides that are the same or different, and insect cleavable peptides that are the same or different.
  • the C-terminal CRIP is operably linked at its C-terminus with a cleavable peptide that is operable to be cleaved in an insect gut environment.
  • the N-terminal CRIP is operably linked at its N-terminus with a cleavable peptide that is operable to be cleaved in an insect gut environment.
  • a cleavable peptide that is operable to be cleaved in an insect gut environment.
  • Some of the available proteases and peptidases found in the insect gut environment are dependent on the life-stage of the insect, as these enzymes are often spatially and temporally expressed.
  • the digestive system of the insect is composed of the alimentary canal and associated glands. Food enters the mouth and is mixed with secretions that may or may not contain digestive proteases and peptidases.
  • the foregut and the hind gut are ectodermal in origin.
  • the foregut serves generally as a storage depot for raw food.
  • the midgut is the site of digestion and absorption of food nutrients.
  • Certain proteases and peptidases in the human gastrointestinal system may include: pepsin, trypsin, chymotrypsin, elastase, carboxypeptidase, aminopeptidase, and dipeptidase.
  • proteases and peptidases found in insect gut environments may include: (1) serine proteases; (2) cysteine proteases; (3) aspartic proteases, and (4) metalloproteases.
  • the two predominant protease classes in the digestive systems of phytophagous insects are the serine and cysteine proteases.
  • Murdock et al. (1987) carried out an elaborate study of the midgut enzymes of various pests belonging to Coleoptera, while Srinivasan et al. (2008) have reported on the midgut enzymes of various pests belonging to Lepidoptera.
  • Serine proteases are known to dominate the larval gut environment and contribute to about 95% of the total digestive activity in Lepidoptera, whereas the Coleopteran species have a wider range of dominant gut proteases, including cysteine proteases.
  • the papain family contains peptidases with a wide variety of activities, including endopeptidases with broad specificity (such as papain), endopeptidases with very narrow specificity (such as glycyl endopeptidases), aminopeptidases, dipeptidyl-peptidase, and peptidases with both endopeptidase and exopeptidase activities (such as cathepsins B and H).
  • proteases found in the midgut of various insects include trypsin-like enzymes, e.g. trypsin and chymotrypsin, pepsin, carboxypeptidase-B and aminotripeptidases.
  • Serine proteases are widely distributed in nearly all animals and microorganisms (Joanitti et al., 2006). In higher organisms, nearly 2% of genes code for these enzymes (Barrette-Ng et al., 2003). Being essentially indispensable to the maintenance and survival of their host organism, serine proteases play key roles in many biological processes.
  • Serine proteases are classically categorized by their substrate specificity, notably by whether the residue at P1: trypsin-like (Lys/Arg preferred at P1), chymotrypsin-like (large hydrophobic residues such as Phe/Tyr/Leu at P1), or elastase-like (small hydrophobic residues such as Ala/Val at P1) (revised by Tyndall et. al.., 2005).
  • Serine proteases are a class of proteolytic enzymes whose central catalytic machinery is composed of three invariant residues, an aspartic acid, a histidine and a uniquely reactive serine, the latter giving rise to their name, the “catalytic triad”.
  • the Asp-His-Ser triad can be found in at least four different structural contexts (Hedstrom, 2002). These four clans of serine proteases are typified by chymotrypsin, subtilisin, carboxypeptidase Y, and Clp protease. The three serine proteases of the chymotrypsin-like clan that have been studied in greatest detail are chymotrypsin, trypsin, and elastase. More recently, serine proteases with novel catalytic triads and dyads have been discovered for their roles in digestion, including Ser-His-Glu, Ser-Lys/His, His-Ser-His, and N-terminal Ser.
  • cysteine proteases One class of well-studied digestive enzymes found in the gut environment of insects is the class of cysteine proteases.
  • cysteine proteases The term “cysteine protease” is intended to describe a protease that possesses a highly reactive thiol group of a cysteine residue at the catalytic site of the enzyme.
  • phytophagous insects and plant parasitic nematodes rely, at least in part, on midgut cysteine proteases for protein digestion.
  • Hemiptera especially squash bugs (Anasa tristis); green stink bug (Acrosternum hilare); Riptortus clavatus; and almost all Coleoptera examined to date, especially, Colorado potato beetle (Leptinotarsa deaemlineata); three-lined potato beetle (Lema trilineata); asparagus beetle (Crioceris asparagi); Mexican bean beetle (Epilachna varivestis); red flour beetle (Triolium castaneum); confused flour beetle (Tribolium confusum); the flea beetles (Chaetocnema spp., Haltica spp., and Epitrix spp.); corn rootworm (Diabrotica Spp.); cowpea weevil (Callosobruchus aculatue); boll weevil (Antonomus grandis); rice weevil (Sitophilus or
  • Aspartic proteases Another class of digestive enzymes is the aspartic proteases.
  • the term “aspartic protease” is intended to describe a protease that possesses two highly reactive aspartic acid residues at the catalytic site of the enzyme and which is most often characterized by its specific inhibition with pepstatin, a low molecular weight inhibitor of nearly all known aspartic proteases.
  • a challenge regarding the expression of heterogeneous polypeptides in transgenic plants is maintaining the desired effect (e.g., insecticidal activity) of the introduced polypeptide upon expression in the host organism; one way to maintain such an effect is to increase the chance of proper protein folding through the use of an operably linked Endoplasmic Reticulum Signal Peptide (ERSP).
  • Another method to maintain the effect of a transgenic protein is to incorporate a Translational Stabilizing Protein (STA).
  • Endoplasmic Reticulum Signal Peptide [00590]
  • the subcellular targeting of a recombinant protein to the ER can be achieved through the use of an ERSP operably linked to said recombinant protein; this allows for the correct assembly and/or folding of such proteins, and the high level accumulation of these recombinant proteins in plants.
  • Exemplary methods concerning the compartmentalization of host proteins into intracellular storage are disclosed in McCormick et al., Proc. Natl. Acad. Sci. USA 96(2):703-708, 1999; Staub et al., Nature Biotechnology 18:333-338, 2000; Conrad et al., Plant Mol.
  • one way to achieve the correct assembly and/or folding of recombinant proteins is to operably link an endoplasmic reticulum signal peptide (ERSP) to the recombinant protein of interest.
  • ESP endoplasmic reticulum signal peptide
  • a peptide comprising an Endoplasmic Reticulum Signal Peptide can be operably linked to a CRIP (designated as ERSP-CRIP), wherein said ERSP is the N-terminal of said peptide, and where the ERSP peptide is between 3 to 60 amino acids in length, between 5 to 50 amino acids in length, between 20 to 30 amino acids in length and or where the peptide is BAAS, or tobacco extensin signal peptide, or a modified tobacco extensin signal peptide, or Jun a 3 signal peptide of Juniperus ashei.
  • ERSP-CRIP Endoplasmic Reticulum Signal Peptide
  • a plant can be transformed with a nucleotide that codes for any of the peptides that are described herein as Endoplasmic Reticulum Signal Peptides (ERSP) and/or a CRIP.
  • a protein comprised of an Endoplasmic Reticulum Signal Peptide (ERSP) can be operably linked to a CRIP, operably linked to an intervening linker peptide (L or Linker), designated as ERSP-L-CRIP, or ERSP-CRIP-L, wherein said ERSP is the N-terminal of said protein, and said L or Linker may be either on the N-terminal side (upstream) of the CRIP or the C-terminal side (downstream) of the CRIP.
  • L or Linker may be either on the N-terminal side (upstream) of the CRIP or the C-terminal side (downstream) of the CRIP.
  • a protein designated as ERSP-L-CRIP, or ERSP-CRIP-L comprising any of the ERSPs or CRIPs described herein and wherein said L can be an uncleavable linker peptide, or a cleavable linker peptide, which may be cleavable in a plant cells during protein expression process or may be cleavable in an insect gut environments and hemolymph environments, and comprised of any of the intervening linker peptide (LINKER) described, or taught by this document including the following sequences: IGER (SEQ ID NO:31), (SEQ ID NO:32), and (SEQ ID NO:33).
  • a protein comprising an Endoplasmic Reticulum Signal Peptide can be operably linked to a CRIP, which is in turn operably linked to a Translational Stabilizing Protein (STA).
  • STA Translational Stabilizing Protein
  • this configuration is designated as ERSP-STA- CRIP or ERSP-CRIP-STA, wherein said ERSP is the N-terminal of said protein and said STA may be either on the N-terminal side (upstream) of the CRIP, or of the C-terminal side (downstream) of the CRIP.
  • a protein designated as ERSP-STA-CRIP or ERSP-CRIP-STA comprising any of the ERSPs or CRIPs described herein, can be operably linked to a STA, for example, any of the translational stabilizing proteins described, or taught by this document including GFP (Green Fluorescent Protein; SEQ ID NO:34; NCBI Accession No. P42212), or Jun a 3, (Juniperus ashei; SEQ ID NO:36; NCBI Accession No. P81295.1).
  • GFP Green Fluorescent Protein
  • SEQ ID NO:34 NCBI Accession No. P42212
  • Jun a 3 Jun a 3
  • Plants can be transiently or stably transfected with the DNA sequence that encodes a CRIP or an insecticidal protein comprising one or more CRIPs and one or more non-CRIP peptides, polypeptides or proteins, for example, using anyone of the transfection methods described above; alternatively, plants can be transfected with a polynucleotide that encodes a CRIP operably linked to an ERSP, LINKER, and/or a STA protein encoding polynucleotide.
  • a transgenic plant or plant genome can be transfected to incorporate the polynucleotide sequence that encodes the Endoplasmic Reticulum Signal Peptide (ERSP); CRIP; and/or intervening linker peptide (LINKER, L), thus causing mRNA transcribed from the heterogeneous DNA to be expressed in the transformed plant.
  • ESP Endoplasmic Reticulum Signal Peptide
  • CRIP CRIP
  • LINKER, L intervening linker peptide
  • the present disclosure may be used for transformation of any plant species, including, but not limited to, monocots and dicots.
  • Crops for which a transgenic approach or PEP would be an especially useful approach include, but are not limited to: alfalfa, cotton, tomato, maize, wheat, corn, sweet corn, lucerne, soybean, sorghum, field pea, linseed, safflower, rapeseed, oil seed rape, rice, soybean, barley, sunflower, trees (including coniferous and deciduous), flowers (including those grown commercially and in greenhouses), field lupins, switchgrass, sugarcane, potatoes, tomatoes, tobacco, crucifers, peppers, sugarbeet, barley, and oilseed rape, Brassica sp., rye, millet, peanuts, sweet potato, cassaya, coffee, coconut, pineapple, citrus trees, cocoa, tea, banana, avocado, fig, guava, mango, olive, papaya, cashew, macadamia, almond, oats, vegetables, ornamentals, and conifers.
  • the CRIP expression open reading frame (ORF) described herein is a polynucleotide sequence that will enable the plant to express mRNA, which in turn will be translated into peptides be expressed, folded properly, and/or accumulated to such an extent that said proteins provide a dose sufficient to inhibit and/or kill one or more pests.
  • an example of a protein CRIP expression ORF can be a cysteine rich insecticidal protein (crip), an “ersp” (i.e., the polynucleotide sequence that encodes the ERSP polypeptide) a “linker” (i.e., the polynucleotide sequence that encodes the LINKER polypeptide), a “sta” (i.e., the polynucleotide sequence that encodes the STA polypeptide), or any combination thereof, and can be described in the following equation format: ersp-sta-(linker i -crip j ) n , or ersp-(crip j -linker i ) n -sta [00597]
  • the foregoing illustrative embodiment of a polynucleotide equation would result in the following protein complex being expressed: ERSP-STA-(LINKERI-CRIPJ)N, containing four possible peptide components with dash signs to separate each
  • the nucleotide component of ersp is a polynucleotide segment encoding a plant endoplasmic reticulum trafficking signal peptide (ERSP).
  • the component of sta is a polynucleotide segment encoding a translation stabilizing protein (STA), which helps the accumulation of the CRIP expressed in plants, however, in some embodiments, the inclusion of sta may not be necessary in the CRIP expression ORF.
  • the component of linker i is a polynucleotide segment encoding an intervening linker peptide (L OR LINKER) to separate the CRIP from other components contained in ORF, and from the translation stabilizing protein.
  • the subscript letter “i” indicates that in some embodiments, different types of linker peptides can be used in the CRIP expression ORF.
  • the component “crip” indicates the polynucleotide segment encoding the CRIP.
  • the subscript “j” indicates different CRIP polynucleotides may be included in the CRIP expression ORF.
  • the CRIP polynucleotide sequence can encode a CRIP with an amino acid substitution, or an amino acid deletion.
  • n indicates that the structure of the nucleotide encoding an intervening linker peptide and a CRIP can be repeated “n” times in the same open reading frame in the same CRIP expression ORF , where “n” can be any integrate number from 1 to 10; “n” can be from 1 to 10, specifically “n” can be 1, 2, 3, 4, or 5, and in some embodiments “n” is 6, 7, 8, 9 or 10.
  • the repeats may contain polynucleotide segments encoding different intervening linkers (LINKER) and different CRIPs.
  • the different polynucleotide segments including the repeats within the same CRIP expression ORF are all within the same translation frame.
  • the inclusion of a sta polynucleotide in the CRIP expression ORF may not be required.
  • an ersp polynucleotide sequence can be directly be linked to the polynucleotide encoding a CRIP variant polynucleotide without a linker.
  • the polynucleotide “crip” encoding the polypeptide “CRIP” can be the polynucleotide sequence that encodes any CRIP as described herein.
  • the “crip” polynucleotide can encode a CRIP including, but not limited to, ACTX peptides (e.g., U-ACTX-Hv1a, U+2-ACTX-Hv1a, rU- ACTX-Hv1a, rU-ACTX-Hv1b, r ⁇ -ACTX-Hv1c, ⁇ -ACTX-Hv1a, and/or ⁇ -ACTX-Hv1a+2); ⁇ -CNTX-Pn1a; U1-agatoxin-Ta1b; TVPs; Av2; Av3; or AVPs.
  • ACTX peptides e.g., U-ACTX-Hv1a, U+2-ACTX-Hv1a, rU- ACTX-Hv1a, rU-ACTX-Hv1b, r ⁇ -ACTX-Hv1c, ⁇ -ACTX-Hv1a, and/or ⁇
  • peptide-IA e.g., an Insecticidal Agent that lends itself to such methods, e.g., a polymer of amino acids, a peptide or a protein
  • Bt toxins e.g., Cry toxins, Cyt toxins, or Vips.
  • the polynucleotide “crip” encoding the polypeptide “CRIP” can be the polynucleotide sequence that encodes any CRIP as described herein, e.g., a CRIP comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% amino acid sequence identity to: a spider peptide having an amino acid sequence as set forth in
  • a CRIP ORF starts with an ersp at its 5’-end.
  • the CRIP For the CRIP to be properly folded and functional when it is expressed from a transgenic plant, it must have an ersp nucleotide fused in frame with the polynucleotide encoding a CRIP.
  • translated ERSP can direct the CRIP being translated to insert into the Endoplasmic Reticulum (ER) of the plant cell by binding with a cellular component called a signal-recognition particle.
  • the ERSP peptide is cleaved by signal peptidase and the CRIP is released into the ER, where the CRIP is properly folded during the post-translation modification process, for example, the formation of disulfide bonds. Without any additional retention protein signals, the protein is transported through the ER to the Golgi apparatus, where it is finally secreted outside the plasma membrane and into the apoplastic space. CRIP can accumulate at apoplastic space efficiently to reach the insecticidal dose in plants. [00601]
  • the ERSP peptide is at the N-terminal region of the plant-translated CRIP complex and the ERSP portion is composed of about 3 to 60 amino acids. In some embodiments it is 5 to 50 amino acids.
  • the ERSP is a signal peptide so called because it directs the transportation of a protein.
  • Signal peptides may also be called targeting signals, signal sequences, transit peptides, or localization signals.
  • the signal peptides for ER trafficking are often 15 to 30 amino acid residues in length and have a tripartite organization, comprised of a core of hydrophobic residues flanked by a positively charged amino terminal and a polar, but uncharged carboxyterminal region.
  • ERSPs are known. It is NOT required that the ERSP be derived from a plant ERSP, non-plant ERSPs will work with the procedures described herein. Many plant ERSPs are however well known and we describe some plant derived ERSPs here.
  • BAAS for example, is derived from the plant, Hordeum vulgare, and has the amino acid sequence as follows: (SEQ ID NO:37) [00603]
  • Plant ERSPs which are selected from the genomic sequence for proteins that are known to be expressed and released into the apoplastic space of plants, include examples such as BAAS, carrot extensin, and tobacco PR1. The following references provide further descriptions, and are incorporated by reference herein in their entirety. De Loose, M. et al. “The extensin signal peptide allows secretion of a heterologous protein from protoplasts” Gene, 99 (1991) 95-100; De Loose, M. et al.
  • the tobacco extensin signal peptide motif is an ERSP (Memelink et al, the Plant Journal, 1993, V4: 1011-1022; see also Pogue GP et al, Plant Biotechnology Journal, 2010, V8: 638-654).
  • a CRIP ORF can have a tobacco extensin signal peptide motif.
  • the CRIP ORF can have an extensin motif according to SEQ ID NO:38.
  • the CRIP ORF can have an extensin motif according to SEQ ID NO:39.
  • a DNA sequence encoding an extensin motif is designed (for example, the DNA sequence shown in SEQ ID NO:40 or SEQ ID NO:41) using oligo extension PCR with four synthetic DNA primers; ends sites such as a restriction site, for example, a Pac I restriction site at the 5’-end, and a 5’-end of a GFP sequence at the 3’-end, can be added using PCR with the extensin DNA sequence serving as a template, and resulting in a fragment; the fragment is used as the forward PCR primer to amplify the DNA sequence encoding a CRIP ORF , for example “gfp-l-CRIP” contained in a pFECT vector, thus producing a CRIP ORF encoding (from N’ to C’ terminal) “ERSP-GFP-L-CRIP” wherein the ERSP is extensin.
  • a restriction site for example, a Pac I restriction site at the 5’-end, and a 5’-end of a GFP sequence at the 3’-end
  • an illustrative expression system can include the FECT expression vectors containing CRIP ORF is transformed into Agrobacterium, GV3101, and the transformed GV3101 is injected into tobacco leaves for transient expression of CRIP ORF.
  • STA Translational stabilizing protein
  • a Translational stabilizing protein (STA) can increase the amount of CRIP in plant tissues.
  • One of the CRIP ORFs is sufficient to express a properly folded CRIP in the transfected plant, but in some embodiments, effective protection of a plant from pest damage may require that the plant expressed CRIP accumulate.
  • a transgenic plant With transfection of a properly constructed CRIP ORF, a transgenic plant can express and accumulate greater amounts of the correctly folded CRIP. When a plant accumulates greater amounts of properly folded CRIP, it can more easily resist, inhibit, and/or kill the pests that attack and eat the plants.
  • One method of increasing the accumulation of a polypeptide in transgenic tissues is through the use of a translational stabilizing protein (STA).
  • STA translational stabilizing protein
  • the translational stabilizing protein can be used to significantly increase the accumulation of CRIP in plant tissue, and thus increase the efficacy of a plant transfected with CRIP with regard to pest resistance.
  • the translational stabilizing protein is a protein with sufficient tertiary structure that it can accumulate in a cell without being targeted by the cellular process of protein degradation.
  • the following equation describes one of the examples of an CRIP ORF that encodes a stabilizing protein fused with U1-agatoxin-Ta1b Variant polynucleotide sequence: ersp-sta-l-crip or ersp-crip-l-sta [00609]
  • the translational stabilizing protein can be a domain of another protein, or it can comprise an entire protein sequence.
  • the translational stabilizing protein can be between 5 and 50 amino acids, 50 to 250 amino acids (e.g., GNA), 250 to 750 amino acids (e.g., chitinase) and 750 to 1500 amino acids (e.g., enhancin).
  • the protein, or protein domain can contain proteins that have no useful characteristics other than translation stabilization, or they can have other useful traits in addition to translational stabilization.
  • One embodiment of the translational stabilizing protein can be a polymer of fusion proteins involving CRIP.
  • a specific example of a translational stabilizing protein is provided here to illustrate the use of a translational stabilizing protein. The example is not intended to limit the disclosure or claims in any way.
  • translational stabilizing proteins are well known in the art, and any proteins of this type could be used as disclosed herein. Procedures for evaluating and testing production of peptides are both known in the art and described herein.
  • One example of one translational stabilizing protein is Green- Fluorescent Protein (GFP) (SEQ ID NO:34; NCBI Accession No. P42212.1).
  • GFP Green- Fluorescent Protein
  • Additional examples of translational stabilizing proteins can be found in the following references, the disclosures of which are incorporated by reference in their entirety: Kramer, K.J. et al.
  • a CRIP ORF can be transformed into a plant, for example, in the tobacco plant, Nicotiana benthamiana, using a CRIP ORF that contains a STA, for example Jun a 3.
  • the mature Jun a 3 is a ⁇ 30 kDa plant defending protein that is also an allergen for some people.
  • Jun a 3 is produced by Juniperus ashei trees and can be used in some embodiments as a translational stabilizing protein (STA).
  • the Jun a 3 amino acid sequence can be the sequence shown in SEQ ID NO:36.
  • the Jun a 3 amino acid sequence can be the sequence shown in SEQ ID NO:42.
  • Linkers [00614] Linker proteins assist in the proper folding of the different motifs composing a CRIP ORF.
  • the CRIP ORF described in this invention also incorporates polynucleotide sequences encoding intervening linker peptides between the polynucleotide sequences encoding the CRIP (crip) and the translational stabilizing protein (sta), or between polynucleotide sequence encoding multiple polynucleotide sequences encoding CRIP, i.e., (l- crip) N or (crip-l) N , if the expression ORF involves multiple CRIP domain expression.
  • the intervening linker peptides (LINKERS or L) separate the different parts of the expressed CRIP complex and help proper folding of the different parts of the complex during the expression process.
  • the intervening linker peptide can be between 1 and 30 amino acids in length. However, it is not necessarily an essential component in the expressed CRIP in plants.
  • a cleavable linker peptide can be designed to the CRIP ORF to release the properly CRIP from the expressed CRIP complex in the transformed plant to improve the protection the CRIP affords the plant with regard to pest damage.
  • intervening linker peptide is the plant cleavable linker peptide. This type of linker peptides can be completely removed from the expressed CRIP ORF complex during plant post-translational modification. Therefore, in some embodiments, the properly folded CRIP linked by this type of intervening linker peptides can be released in the plant cells from the expressed CRIP ORF complex during post-translational modification in the plant.
  • Another type of the cleavable intervening linker peptide is not cleavable during the expression process in plants. However, it has a protease cleavage site specific to serine, threonine, cysteine, aspartate proteases or metalloproteases.
  • the type of cleavable linker peptide can be digested by proteases found in the insect and lepidopteran gut environment and/or the insect hemolymph and lepidopteran hemolymph environment to release the CRIP in the insect gut or hemolymph.
  • proteases found in the insect and lepidopteran gut environment and/or the insect hemolymph and lepidopteran hemolymph environment to release the CRIP in the insect gut or hemolymph.
  • the CRIP ORF can contain a cleavable type of intervening linker, for example, the type listed in SEQ ID NO:31, having the amino acid code of “IGER” (SEQ ID NO:31).
  • the molecular weight of this intervening linker or LINKER is 473.53 Daltons.
  • the intervening linker peptide can also be one without any type of protease cleavage site, i.e. an uncleavable intervening linker peptide, for example, the linker “ETMFKHGL” (SEQ ID NO:33).
  • the CRIP-insecticidal protein can have two or more cleavable peptides, wherein the insecticidal protein comprises an insect cleavable linker (L), the insect cleavable linker being fused in frame with a construct comprising (CRIP-L) n , wherein “n” is an integer ranging from 1 to 200, or from 1 to 100, or from 1 to 10.
  • the CRIP-insecticidal protein comprises an endoplasmic reticulum signal peptide (ERSP) operably linked with a CRIP, which is operably linked with an insect cleavable linker (L) and/or a repeat construct (L-CRIP) n or (CRIP-L) n , wherein n is an integer ranging from 1 to 200, or from 1 to 100, or from 1 to 10.
  • SRP endoplasmic reticulum signal peptide
  • L insect cleavable linker
  • L-CRIP repeat construct
  • CRIP-L CRIP-L
  • a protein comprising an Endoplasmic Reticulum Signal Peptide can be operably linked to a CRIP and an intervening linker peptide (L or Linker); such a construct is designated as ERSP-L-CRIP, or ERSP-CRIP-L, wherein said ERSP is the N-terminal of said protein, and said L or Linker may be either on the N-terminal side (upstream) of the CRIP, or the C-terminal side (downstream) of the CRIP.
  • a protein designated as ERSP-L-CRIP, or ERSP-CRIP-L, comprising any of the ERSPs or CRIPs described herein, can have a Linker “L” that can be an uncleavable linker peptide, or a cleavable linker peptide, and which may be cleavable in a plant cells during protein expression process, or may be cleavable in an insect gut environment and/or hemolymph environment.
  • L can be an uncleavable linker peptide, or a cleavable linker peptide, and which may be cleavable in a plant cells during protein expression process, or may be cleavable in an insect gut environment and/or hemolymph environment.
  • linker peptides can be found in the following references, which are incorporated by reference herein in their entirety: A plant expressed serine proteinase inhibitor precursor was found to contain five homogeneous protein inhibitors separated by six same linker peptides in Heath et al. “Characterization of the protease processing sites in a multidomain proteinase inhibitor precursor from Nicotiana alata” European Journal of Biochemistry, 1995; 230: 250-257. A comparison of the folding behavior of green fluorescent proteins through six different linkers is explored in Chang, H.C. et al.
  • TMV tobacco mosaic virus
  • TMOF trypsin-modulating oostatic factor
  • CRIP ORF refers to a nucleotide encoding a CRIP, and/or one or more stabilizing proteins, secretory signals, or target directing signals, for example, ERSP or STA, and is defined as the nucleotides in the ORF that has the ability to be translated.
  • a “CRIP ORF diagram” refers to the composition of one or more CRIP ORFs, as written out in diagram or equation form.
  • a “CRIP ORF diagram” can be written out as using acronyms or short-hand references to the DNA segments contained within the expression ORF. Accordingly, in one example, a “CRIP ORF diagram” may describe the polynucleotide segments encoding the ERSP, LINKER, STA, and CRIP, by diagramming in equation form the DNA segments as “ersp” (i.e., the polynucleotide sequence that encodes the ERSP polypeptide); “linker” or “L” (i.e., the polynucleotide sequence that encodes the LINKER polypeptide); “sta” (i.e., the polynucleotide sequence that encodes the STA polypeptide), and “crip” (i.e., the polynucleotide sequence encoding a CRIP), respectively.
  • ersp i.e., the polynucleotide sequence that encodes the ERSP polypeptide
  • linker or “L” i.e
  • CRIP ORF diagram An example of a CRIP ORF diagram is “ersp-sta-(linker i -crip j )N,” or “ersp-(crip j -linkeri)N-sta” and/or any combination of the DNA segments thereof.
  • the following equations describe two examples of a CRIP ORF that encodes an ERSP, a STA, a linker, and a CRIP: ersp-sta-l-crip or ersp-crip-l-sta
  • the CRIP open reading frame (ORF) described herein is a polynucleotide sequence that will enable the plant to express mRNA, which in turn will be translated into peptides that will folded properly, and/or accumulated to such an extent that said proteins provide a dose sufficient to inhibit and/or kill one or more pests.
  • an example of a protein CRIP ORF can be a polynucleotide encoding a CRIP (crip), an “ersp” (i.e., the polynucleotide sequence that encodes the ERSP polypeptide) a “linker” (i.e., the polynucleotide sequence that encodes the LINKER polypeptide), a “sta” (i.e., the polynucleotide sequence that encodes the STA polypeptide), or any combination thereof, and can be described in the following equation format: ersp-sta-(linker i -crip j ) n , or ersp-(crip j -linker i ) n -sta [00626]
  • the foregoing illustrative embodiment of a polynucleotide equation would result in the following protein complex being expressed: ERSP-STA-(LINKERI-CRIPJ)N, containing four possible peptide components with dash
  • the nucleotide component of ersp is a polynucleotide segment encoding a plant endoplasmic reticulum trafficking signal peptide (ERSP).
  • the component of sta is a polynucleotide segment encoding a translation stabilizing protein (STA), which helps the accumulation of the CRIP expressed in plants, however, in some embodiments, the inclusion of sta may not be necessary in the CRIP ORF.
  • the component of linker i is a polynucleotide segment encoding an intervening linker peptide (L OR LINKER) to separate the CRIP from other components contained in ORF, and from the translation stabilizing protein.
  • the subscript letter “i” indicates that in some embodiments, different types of linker peptides can be used in the CRIP ORF.
  • the component “crip” indicates the polynucleotide segment encoding the CRIP.
  • the subscript “j” indicates different polynucleotides may be included in the CRIP ORF.
  • the polynucleotide sequence can encode a CRIP with a different amino acid substitution.
  • n indicates that the structure of the nucleotide encoding an intervening linker peptide and a CRIP can be repeated “n” times in the same open reading frame in the same CRIP ORF , where “n” can be any integrate number from 1 to 10; “n” can be from 1 to 10, specifically “n” can be 1, 2, 3, 4, or 5, and in some embodiments “n” is 6, 7, 8, 9 or 10.
  • the repeats may contain polynucleotide segments encoding different intervening linkers (LINKER) and different CRIPs. The different polynucleotide segments including the repeats within the same CRIP ORF are all within the same translation frame.
  • the inclusion of a sta polynucleotide in the CRIP ORF may not be required.
  • an ersp polynucleotide sequence can be directly be linked to the polynucleotide encoding a CRIP variant polynucleotide without a linker.
  • the polynucleotide “crip” encoding the polypeptide “CRIP” can be the polynucleotide sequence that encodes any CRIP as described herein, e.g., a CRIP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8%
  • a polynucleotide is operable to encode a CRIP- insecticidal protein having the following CRIP construct orientation and/or arrangement: ERSP-CRIP; ERSP-(CRIP) N ; ERSP-CRIP-L; ERSP-(CRIP) N -L; ERSP-(CRIP-L) N ; ERSP-L- CRIP; ERSP-L-(CRIP) N ; ERSP-(L-CRIP) N ; ERSP-STA-CRIP; ERSP-STA-(CRIP) N ; ERSP- CRIP-STA; ERSP-(CRIP)N-STA; ERSP-(STA-CRIP)N; ERSP-(CRIP-STA)N; ERSP-L- CRIP-STA; ERSP-L-(CRIP-STA)N; ERSP-L-CRIP; ERSP-L-(CRIP-STA)N; ERSP-L-
  • any of the CRIP ORFs and/or CRIP constructs described herein can be produced recombinantly, e.g., in some embodiments, any of the CRIP ORFs and/or CRIP constructs described herein can be produced in cell culture, e.g., by a yeast cell.
  • any of the aforementioned methods, and/or any of the methods described herein, can be used to incorporate into a plant or a plant part thereof, one or more polynucleotides operable to express any one or more of the CRIPs or CRIP-insecticidal proteins as described herein; e.g., one or more CRIPs or CRIP-insecticidal protein having the amino acid sequence of SEQ ID NOs: 2-15, 49-53, or 77-110, which are likewise described herein.
  • the present disclosure may be used for transformation of any plant species, including, but not limited to, monocots and dicots.
  • Crops for which a transgenic approach or PEP would be an especially useful approach include, but are not limited to: alfalfa, cotton, tomato, maize, wheat, corn, sweet corn, lucerne, soybean, sorghum, field pea, linseed, safflower, rapeseed, oil seed rape, rice, soybean, barley, sunflower, trees (including coniferous and deciduous), flowers (including those grown commercially and in greenhouses), field lupins, switchgrass, sugarcane, potatoes, tomatoes, tobacco, crucifers, peppers, sugarbeet, barley, and oilseed rape, Brassica sp., rye, millet, peanuts, sweet potato, cassaya, coffee, coconut, pineapple, citrus trees, cocoa, tea, banana, avocado, fig, guava, mango, olive, papaya, cashew, macadamia, almond, oats, vegetables, ornamentals, and conifers.
  • the CRIP ORFs and CRIP constructs described above and herein can be cloned into any plant expression vector for the CRIP to be expression in plants, either transiently or stably.
  • Transient plant expression systems can be used to promptly optimize the structure of the CRIP ORF for some specific CRIP expression in plants, including the necessity of some components, codon optimization of some components, optimization of the order of each component, etc.
  • a transient plant expression vector is often derived from a plant virus genome. Plant virus vectors provide advantages in quick and high level of foreign gene expression in plant due to the infection nature of plant viruses.
  • the full length of the plant viral genome can be used as a vector, but often a viral component is deleted, for example the coat protein, and transgenic ORFs are subcloned in that place.
  • the CRIP ORF can be subcloned into such a site to create a viral vector.
  • These viral vectors can be introduced into plant mechanically since they are infectious themselves, for example through plant wound, spray-on etc. They can also be transfected into plants via agroinfection, by cloning the virus vector into the T-DNA of the crown gall bacterium, Agrobacterium tumefaciens, or the hairy root bacterium, Agrobacterium rhizogenes.
  • the expression of the CRIP in this vector is controlled by the replication of the RNA virus, and the virus translation to mRNA for replication is controlled by a strong viral promoter, for example, 35S promoter from Cauliflower mosaic virus.
  • Viral vectors with CRIP ORF are usually cloned into T-DNA region in a binary vector that can replicate itself in both E. coli strains and Agrobacterium strains.
  • the transient transfection of a plant can be done by infiltration of the plant leaves with the Agrobacterium cells which contain the viral vector for CRIP expression. In the transient transformed plant, it is common for the foreign protein expression to be ceased in a short period of time due to the post-transcriptional gene silencing (PTGS).
  • PTGS post-transcriptional gene silencing
  • transient transfection of plants can be achieved by recombining a polynucleotide encoding a CRIP with any one of the readily available vectors (see above), and confirmed, using a marker or signal (e.g., GFP emission).
  • a marker or signal e.g., GFP emission
  • a transiently transfected plant can be created by recombining a polynucleotide encoding a CRIP with a DNA encoding a GFP-Hybrid fusion protein in a vector, and transfection said vector into a plant (e.g., tobacco) using different FECT vectors designed for targeted expression.
  • a polynucleotide encoding a CRIP can be recombined with a pFECT vector for APO (apoplast localization) accumulation; a pFECT vector for CYTO (cytoplasm localization) accumulation; or pFECT with ersp vector for ER (endoplasm reticulum localization) accumulation.
  • An exemplary transient plant transformation strategy is agroinfection using a plant viral vector due to its high efficiency, ease, and low cost.
  • a tobacco mosaic virus overexpression system (see TRBO, Lindbo JA, Plant Physiology, 2007, V145: 1232-1240) can be used to transiently transform plants with CRIP.
  • the TRBO DNA vector has a T-DNA region for agroinfection, which contains a CaMV 35S promoter that drives expression of the tobacco mosaic virus RNA without the gene encoding the viral coating protein.
  • this system uses the “disarmed” virus genome, therefore viral plant to plant transmission can be effectively prevented.
  • the FECT viral transient plant expression system can be used to transiently transform plants with CRIP (see Liu Z & Kearney CM, BMC Biotechnology, 2010, 10:88).
  • the FECT vector contains a T-DNA region for agroinfection, which contains a CaMV 35S promoter that drives the expression of the foxtail mosaic virus RNA without the genes encoding the viral coating protein and the triple gene block.
  • this system uses the “disarmed” virus genome, therefore viral plant to plant transmission can be effectively prevented.
  • the FECT expression system additionally needs to co-express P19, a RNA silencing suppressor protein from tomato bushy stunt virus, to prevent the post-transcriptional gene silencing (PTGS) of the introduced T-DNA.
  • P19 a RNA silencing suppressor protein from tomato bushy stunt virus
  • PTGS post-transcriptional gene silencing
  • the CRIP ORF can be designed to encode a series of translationally fused structural motifs that can be described as follows: N’-ERSP-STA-L- CRIP-C’ wherein the “N’” and “C’” indicating the N-terminal and C-terminal amino acids, respectively, and the ERSP motif can be the Barley Alpha-Amylase Signal peptide (BAAS) (SEQ ID NO:37); the stabilizing protein (STA) can be GFP (SEQ ID NO:34); the linker peptide “L” can be IGER (SEQ ID NO:31)
  • the ersp-sta-l-CRIP ORF can chemically synthesized to include restrictions sites, for example a Pac I restriction site at its 5’-end, and an Avr II restriction site at its 3’-end.
  • the CRIP ORF can be cloned into the Pac I and Avr II restriction sites of a FECT expression vector (pFECT) to create an U1-agatoxin-Ta1b variant expression vector for the FECT transient plant expression system (pFECT-CRIP).
  • pFECT FECT expression vector
  • pFECT-CRIP FECT transient plant expression system
  • some embodiments may have a FECT vector expressing the RNA silencing suppressor protein P19 (pFECT-P19) generated for co-transformation.
  • a U1-agatoxin-Ta1b variant expression vector can be recombined for use in a TRBO transient plant expression system, for example, by performing a routine PCR procedure and adding a Not I restriction site to the 3’-end of the CRIP ORF described above, and then cloning the CRIP ORF into Pac I and Not I restriction sites of the TRBO expression vector (pTRBO-CRIP).
  • an Agrobacterium tumefaciens strain for example, commercially available GV3101 cells, can be used for the transient expression of a CRIP ORF in a plant tissue (e.g., tobacco leaves) using one or more transient expression systems, for example, the FECT and TRBO expression systems.
  • An exemplary illustration of such a transient transfection protocol includes the following: an overnight culture of GV3101 can be used to inoculate 200 mL Luria-Bertani (LB) medium; the cells can be allowed to grow to log phase with OD600 between 0.5 and 0.8; the cells can then be pelleted by centrifugation at 5000 rpm for 10 minutes at 4°C; cells can then be washed once with 10 mL pre-chilled TE buffer (Tris-HCl 10 mM, EDTA 1mM, pH8.0), and then resuspended into 20 mL LB medium; GV3101 cell resuspension can then be aliquoted in 250 ⁇ L fractions into 1.5 mL microtubes; aliquots can then be snap-frozen in liquid nitrogen and stored at -80°C freezer for future transformation.
  • LB Luria-Bertani
  • the pFECT-CRIP and pTRBO-CRIP vectors can then transformed into the competent GV3101 cells using a freeze-thaw method as follows: the stored competent GV3101 cells are thawed on ice and mixed with 1 to 5 ⁇ g pure DNA (pFECT- CRIP or pTRBO-CRIP vector). The cell-DNA mixture is kept on ice for 5 minutes, transferred to -80°C for 5 minutes, and incubated in a 37°C water bath for 5 minutes. The freeze-thaw treated cells are then diluted into 1 mL LB medium and shaken on a rocking table for 2 to 4 hours at room temperature.
  • a 200 ⁇ L aliquot of the cell-DNA mixture is then spread onto LB agar plates with the appropriate antibiotics (10 ⁇ g/mL rifampicin, 25 ⁇ g/mL gentamycin, and 50 ⁇ g/mL kanamycin can be used for both pFECT-CRIP transformation and pTRBO-CRIP transformation) and incubated at 28°C for two days. Resulting transformed colonies are then picked and cultured in 6 mL aliquots of LB medium with the appropriate antibiotics for transformed DNA analysis and making glycerol stocks of the transformed GV3101 cells.
  • the appropriate antibiotics 10 ⁇ g/mL rifampicin, 25 ⁇ g/mL gentamycin, and 50 ⁇ g/mL kanamycin can be used for both pFECT-CRIP transformation and pTRBO-CRIP transformation
  • the transient transformation of plant tissues can be performed using leaf injection with a 3-mL syringe without needle.
  • the transformed GV3101 cells are streaked onto an LB plate with the appropriate antibiotics (as described above) and incubated at 28°C for two days.
  • a colony of transformed GV3101 cells are inoculated to 5 ml of LB-MESA medium (LB media supplemented with 10 mM MES, and 20 ⁇ M acetosyringone) and the same antibiotics described above, and grown overnight at 28°C.
  • the cells of the overnight culture are collected by centrifugation at 5000 rpm for 10 minutes and resuspended in the induction medium (10 mM MES, 10 mM MgCl 2 , 100 ⁇ M acetosyringone) at a final OD600 of 1.0.
  • the cells are then incubated in the induction medium for 2 hours to overnight at room temperature and are then ready for transient transformation of tobacco leaves.
  • the treated cells can be infiltrated into the underside of attached leaves of Nicotiana benthamiana plants by injection, using a 3-mL syringe without a needle attached.
  • the transient transformation can be accomplished by transfecting one population of GV3101 cells with pFECT-CRIP or pTRBO-CRIP and another population with pFECT-P19, mixing the two cell populations together in equal amounts for infiltration of tobacco leaves by injection with a 3-mL syringe.
  • Stable integration of polynucleotide operable to encode CRIP is also possible with the present disclosure, for example, the CRIP ORF can also be integrated into plant genome using stable plant transformation technology, and therefore CRIPs can be stably expressed in plants and protect the transformed plants from generation to generation.
  • the CRIP expression vector can be circular or linear.
  • the CRIP ORF, the CRIP expression cassette, and/or the vector with polynucleotide encoding an CRIP for stable plant transformation should be carefully designed for optimal expression in plants based on what is known to those having ordinary skill in the art, and/or by using predictive vector design tools such as Gene Designer 2.0 (Atum Bio); VectorBuilder (Cyagen); SnapGene® viewer; GeneArt TM Plasmid Construction Service (Thermo-Fisher Scientific); and/or other commercially available plasmid design services. See Tolmachov, Designing plasmid vectors. Methods Mol Biol.2009; 542:117-29.
  • the expression of CRIP is usually controlled by a promoter that promotes transcription in some, or all the cells of the transgenic plant.
  • the promoter can be a strong plant viral promoter, for example, the constitutive 35S promoter from Cauliflower Mosaic Virus (CaMV); it also can be a strong plant promoter, for example, the hydroperoxide lyase promoter (pHPL) from Arabidopsis thaliana; the Glycine max polyubiquitin (Gmubi) promoter from soybean; the ubiquitin promoters from different plant species (rice, corn, potato, etc.), etc.
  • a plant transcriptional terminator often occurs after the stop codon of the ORF to halt the RNA polymerase and transcription of the mRNA.
  • a reporter gene can be included in the CRIP expression vector, for example, beta-glucuronidase gene (GUS) for GUS straining assay, green fluorescent protein (GFP) gene for green fluorescence detection under UV light, etc.
  • GUS beta-glucuronidase gene
  • GFP green fluorescent protein
  • a selection marker gene is usually included in the CRIP expression vector.
  • the marker gene expression product can provide the transformed plant with resistance to specific antibiotics, for example, kanamycin, hygromycin, etc., or specific herbicide, for example, glyphosate etc. If agroinfection technology is adopted for plant transformation, T-DNA left border and right border sequences are also included in the CRIP expression vector to transport the T-DNA portion into the plant.
  • the constructed CRIP expression vector can be transfected into plant cells or tissues using many transfection technologies.
  • Agroinfection is a very popular way to transform a plant using an Agrobacterium tumefaciens strain or an Agrobacterium rhizogenes strain.
  • Particle bombardment also called Gene Gun, or Biolistics
  • Other less common transfection methods include tissue electroporation, silicon carbide whiskers, direct injection of DNA, etc.
  • Evaluation of a transformed plant can be accomplished at the DNA level, RNA level and protein level.
  • a stably transformed plant can be evaluated at all of these levels and a transiently transformed plant is usually only evaluated at protein level.
  • the genomic DNA can be extracted from the stably transformed plant tissues for and analyzed using PCR or Southern blot.
  • the expression of the CRIP in the stably transformed plant can be evaluated at the RNA level, for example, by analyzing total mRNA extracted from the transformed plant tissues using northern blot or RT-PCR.
  • the expression of the CRIP in the transformed plant can also be evaluated in protein level directly. There are many ways to evaluate expression of CRIP in a transformed plant.
  • a reporter gene assay can be performed, for example, in some embodiments a GUS straining assay for GUS reporter gene expression, a green fluorescence detection assay for GFP reporter gene expression, a luciferase assay for luciferase reporter gene expression, and/or other reporter techniques may be employed.
  • total protein can be extracted from the transformed plant tissues for the direct evaluation of the expression of the CRIP using a Bradford assay to evaluate the total protein level in the sample.
  • a plant, plant tissue, plant cell, plant seed, or part thereof of the present invention can comprise one or more CRIPs, or a polynucleotide encoding the same.
  • a plant, plant tissue, plant cell, plant seed, or part thereof of the present invention can comprise one or more CRIPs, or a polynucleotide encoding the same, wherein said CRIP is any CRIP as described herein.
  • a plant, plant tissue, plant cell, plant seed, or part thereof can comprise a CRIP including, but not limited to, one or more of the following: ACTX peptides (e.g., U- ACTX-Hv1a, U+2-ACTX-Hv1a, rU-ACTX-Hv1a, rU-ACTX-Hv1b, r ⁇ -ACTX-Hv1c, ⁇ - ACTX-Hv1a, and/or ⁇ -ACTX-Hv1a+2); ⁇ -CNTX-Pn1a; U1-agatoxin-Ta1b; TVPs; Av2; Av3; or AVPs.
  • ACTX peptides e.g., U- ACTX-Hv1a, U+2-ACTX-Hv1a, rU-ACTX-Hv1a, rU-ACTX-Hv1b, r ⁇ -ACTX-Hv1c, ⁇ -
  • the methods and techniques can be used to create a plant, plant tissue, plant cell, plant seed, or part thereof, comprising a peptide-IA (e.g., an Insecticidal Agent that lends itself to such methods, e.g., a polymer, a peptide or a protein) such as Bt toxins (e.g., Cry toxins, Cyt toxins, or Vips).
  • a peptide-IA e.g., an Insecticidal Agent that lends itself to such methods, e.g., a polymer, a peptide or a protein
  • Bt toxins e.g., Cry toxins, Cyt toxins, or Vips.
  • a plant, plant tissue, plant cell, plant seed, or part thereof of the present invention can comprise one or more CRIPs, or a polynucleotide encoding the same, wherein said CRIP may comprise an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% amino acid sequence identity to: a spider peptide having an amino acid sequence as set forth in any one of SEQ ID NOs:
  • PCR analysis is a rapid method to screen transformed cells, tissue or shoots for the presence of incorporated gene at the earlier stage before transplanting into the soil (Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). PCR is carried out using oligonucleotide primers specific to the gene of interest or Agrobacterium vector background, etc.
  • Plant transformation may be confirmed by Southern blot analysis of genomic DNA (Sambrook and Russell, 2001, supra). In general, total DNA is extracted from the transformed plant, digested with appropriate restriction enzymes, fractionated in an agarose gel and transferred to a nitrocellulose or nylon membrane. The membrane or "blot" is then probed with, for example, radiolabeled 32 P target DNA fragment to confirm the integration of introduced gene into the plant genome according to standard techniques (Sambrook and Russell, 2001, supra).
  • RNA is isolated from specific tissues of transformed plant, fractionated in a formaldehyde agarose gel, and blotted onto a nylon filter according to standard procedures that are routinely used in the art (Sambrook and Russell, 2001, supra). Expression of RNA encoded by the polynucleotide encoding a CRIP is then tested by hybridizing the filter to a radioactive probe derived from a CRIP, by methods known in the art (Sambrook and Russell, 2001, supra).
  • genes have been reported (Stalker et al. (1985) J. Biol. Chem.263:6310- 6314 (bromoxynil resistance nitrilase gene); and Sathasivan et al. (1990) Nucl. Acids Res. 18:2188 (AHAS imidazolinone resistance gene). Additionally, the genes disclosed herein are useful as markers to assess transformation of bacterial, yeast, or plant cells. Methods for detecting the presence of a transgene in a plant, plant organ (e.g., leaves, stems, roots, etc.), seed, plant cell, propagule, embryo or progeny of the same are well known in the art. In one embodiment, the presence of the transgene is detected by testing for pesticidal activity.
  • Fertile plants expressing a CRIP and/or U1-agatoxin-Ta1b variant polynucleotide may be tested for pesticidal activity, and the plants showing optimal activity selected for further breeding. Methods are available in the art to assay for pest activity. Generally, the protein is mixed and used in feeding assays. See, for example Marrone et al. (1985) J. of Economic Entomology 78:290-293. [00659] In some embodiments, evaluating the success of a transient transfection procedure can be determined based on the expression of a reporter gene, for example, GFP. In some embodiments, GFP can be detected under U.V. light in tobacco leaves transformed with the FECT and/or TRBO vectors.
  • a reporter gene for example, GFP.
  • GFP can be detected under U.V. light in tobacco leaves transformed with the FECT and/or TRBO vectors.
  • CRIP expression can be quantitatively evaluated in a plant (e.g., tobacco).
  • a plant e.g., tobacco
  • An exemplary procedure that illustrates CRIP quantification in a tobacco plant is as follows: 100 mg disks of transformed leaf tissue is collected by punching leaves with the large opening of a 1000 ⁇ L pipette tip. The collected leaf tissue is place into a 2 mL microtube with 5/32” diameter stainless steel grinding balls, and frozen in -80°C for 1 hour, and then homogenized using a Troemner-Talboys High Throughput Homogenizer.
  • TSP-SE1 extraction solutions sodium phosphate solution 50 mM, 1:100 diluted protease inhibitor cocktail, EDTA 1mM, DIECA 10mM, PVPP 8%, pH 7.0
  • the microtube is then left still at room temperature for 15 minutes and then centrifuged at 16,000 g for 15 minutes at 4°C; 100 ⁇ L of the resulting supernatant is taken and loaded into pre-Sephadex G-50-packed column in 0.45 ⁇ m Millipore MultiScreen filter microtiter plate with empty receiving Costar microtiter plate on bottom.
  • the microtiter plates are then centrifuged at 800 g for 2 minutes at 4°C.
  • the resulting filtrate solution herein called total soluble protein extract (TSP extract) of the tobacco leaves.
  • TSP extract total soluble protein extract
  • the total soluble protein concentration of the TSP extract can be estimated using Pierce Coomassie Plus protein assay.
  • BSA protein standards with known concentrations can be used to generate a protein quantification standard curve. For example, 2 ⁇ L of each TSP extract can be mixed into 200 ⁇ L of the chromogenic reagent (CPPA reagent) of the Coomassie Plus protein assay kits and incubated for 10 minutes.
  • CPPA reagent chromogenic reagent
  • the chromogenic reaction can then be evaluated by reading OD595 using a SpectroMax-M2 plate reader using SoftMax Pro as control software.
  • concentrations of total soluble proteins can be about 0.788 ⁇ 0.20 ⁇ g/ ⁇ L or about 0.533 ⁇ 0.03 ⁇ g/ ⁇ L in the TSP extract from plants transformed via FECT and TRBO, respectively, and the results can be used to calculate the percentage of the expressed U1-agatoxin-Ta1b Variant peptide in the TSP (%TSP) for the iELISA assay [00662]
  • an indirect ELISA (iELISA) assay can be used to quantitatively evaluate the CRIP content in the tobacco leaves transiently transformed with the FECT and/or TRBO expression systems.
  • the expressed CRIP can be detected by iELISA at about 3.09 ⁇ 1.83 ng/ ⁇ L in the leaf TSP extracts from the FECT transformed tobacco; and about 3.56 ⁇ 0.74 ng/ ⁇ L in the leaf TSP extract from the TRBO transformed tobacco.
  • the expressed CRIP can be about 0.40% total soluble protein (%TSP) for FECT transformed plants and about 0.67% TSP in TRBO transformed plants.
  • IAs Insecticidal Agents
  • IAs are chemical substances, molecules, nucleotides, polynucleotides, peptides, polypeptides, proteins, toxins, toxicants, poisons, insecticides, pesticides, organic compounds, inorganic compounds, prokaryote organisms, or eukaryote organisms (and the agents produced from said prokaryote or eukaryote organisms), that possess at least some insecticidal activity.
  • an IA can be any one or more chemical substances, molecules, nucleotides, polynucleotides, peptides, polypeptides, proteins, poisons, insecticides, pesticides, organic compounds, inorganic compounds, or combinations thereof, that exhibit insecticidal activity.
  • an IA can be a prokaryote organism, eukaryote organism, or the agents produced therefrom, that exhibit insecticidal activity.
  • an IA includes, but is not limited to, members selected from the categories of: RNAi; Stomach poisons; Inhibitors of chitin biosynthesis type 0; Inhibitors of chitin biosynthesis, type 1; Insect viruses; Compounds isolated from Azadirachta indica; Compounds with unknown MOAs; Bacteria (and products therefrom); Fungi (and products therefrom); Nematodes (and products therefrom); Botanical essences; Mechanical disruptors; Fluorescent brighteners; Silica nanospheres; Chitinases; Lectins; Membrane Attack Complex/Perforin (MACPF) proteins; Plant virus coat protein-toxin fusions; Glycan binding domain/toxin fusion proteins; Acetylcholinesterase (AchE) inhibitors; GABA-gated chloride channel blockers; Sodium channel modulators; Nicotinic acetylcholine receptor (nAchR) Competitive Modulators; Nicot
  • an Insecticidal Agent can be selected from the following: RNAi: such as dsRNA (e.g., WupA dsRNA); Stomach poisons: e.g., arsenicals such as “Paris green” or copper acetoarsenite, lead arsenate, calcium arsenate; fluorine compounds (e.g., sodium fluoride); borates (e.g., borax, boric acid, disodium octaborate, sodium borate, sodium metaborate, sodium tetraborate decahydrate, boron oxide, boron carbide, boron nitride, boron tribromide, boron trichloride, or boron trifluoride); Inhibitors of chitin biosynthesis type 0: e.g., benzoylureas (e.g., bistrifluron, chlorfluazuron, diflubenz
  • Bacillus thuringiensis toxins e.g., parasporal crystal toxins (e.g., ⁇ - endotoxins such as Cry toxins, Cyt toxins); or secreted protein (e.g., vegetative insecticidal proteins (Vips), secreted insecticidal proteins (Sips), Bin-like family proteins, or ETX_MTX2-family proteins); Fungi: including parts and/or products thereof, e.g., Ascomycete fungi, such as a fungi in the Cordycipitaceae family (e.g., a Beauveria bassiana or Cordyceps bassiana, and/or the toxins therefrom); Metarhizium anisopliae (
  • an Insecticidal Agent can be selected from the following group: Acetylcholinesterase (AchE) inhibitors: e.g., carbamates (e.g., alanycarb, aldicarb, bendiocarb, benfuracarb, butocarboxim, butoxycarboxim, carbaryl, carbofuran, carbosulfan, ethiofencarb, fenobucarb, formetanate, furathiocarb, isoprocarb, methiocarb, methomyl, metolcarb, oxamyl, pirimicarb, propoxur, thiodicarb, thiofanox, triazamate, trimethacarb, xmc, and xylylcarb); and organophosphates (e.g., acephate, azamethiphos, azinphos-ethyl,
  • an IA can be a nucleotide, polynucleotide, gene, peptide, polypeptide, protein, or enzyme.
  • an IA can be expressed in plants.
  • an IA operable to be expressed in a plant, plant tissue, plant cell, plant seed, or plant part thereof can include one or more of the following: nucleotides, peptides, polypeptides, and/or proteins isolated from the organisms known as Bacillus thuringiensis var. israelensis, Bacillus thuringiensis var. aizawai, Bacillus thuringiensis var.
  • israeltaki Bacillus thuringiensis var. tenebrionensis, and/or Bacillus sphaericus; chitinases; Galanthus nivalis agglutinin; WupA dsRNA; TIC4670 Beta pore forming protein; AfIP-1A/1B, PIP- 72Aa, luteovirus CP-toxin fusion; chitinase glycan binding domain from Yersinia entomophaga MH96; glycan binding domain from snowdrop lectin fused to a toxin; glycan binding domain from plant Luteovirus fused to toxin; and glycan binding domain from insect Parvoviridae or Baculoviridae coat protein viruses fused to toxin.
  • Entomopathogenic fungi are fungi that can an act as a parasite and/or disease of insect and/or invertebrates. As their name implies, entomopathogenic fungi are eukaryote organisms, having a nucleus clearly defined by a membrane. Entomopathogenic fungi can be a single-cell organism (i.e., unicellular), such as in yeasts; alternatively, they can be formed multicellularly via filamentous units known as hyphae, forming mycelium. Hyphae are formed by single-nucleate or multi-nucleate segments, separated by transverse walls.
  • Fungi reproduction units are known as spores or conidia. As it pertains to entomopathogenic fungi, target insects are usually infected by these reproductive units. Generally, the infection of insects by entomopathogenic fungi is usually divided into three steps: (1) adhesion and spore germination in the insect’s cuticle; (2) penetration within the insect’s hemocele; and (3) fungi development, generally ending with the insect’s death. See Tanada, Y., & Kaya, H. K. (1993), Insect Pathology. San Diego. Academic Press.
  • an IA can be an entomopathogenic fungi, or product derived therefrom, for example, hyphae, spores or reproductive structures.
  • an IA can be a peptide, protein, or toxin produced from an entomopathogenic fungi.
  • an IA can be an Ascomycete fungal toxin.
  • an IA can be a Cordycipitaceae family fungal toxin.
  • an IA can be is a Akanthomyces toxin; a Ascopolyporus toxin; a Beauveria toxin; a Beejasamuha toxin; a Cordyceps toxin; a Coremiopsis toxin; a Engyodontium toxin; a Gibellula toxin; a Hyperdermium toxin; a Insecticola toxin; a Isaria toxin; a Lecanicillium toxin; a Microhilum toxin; a Phytocordyceps toxin; a Pseudogibellula toxin; a Rotiferophthora toxin;
  • an IA can be an organism or toxin therefrom, selected from the following genera: Beauveria; Metarhizium; Paecilomyces; Lecanicillium; Nomuraea; Isaria; Hirsutella; Sorosporella; Aspergillus; Cordiceps; Entomophthora; Zoophthora; Pandora; Entomophaga; Conidiobolus and Basidiobolus.
  • an IA can be a Beauveria toxin.
  • an IA can be: a Beauveria alba toxin; a Beauveria amorpha toxin; a Beauveria arenaria toxin; a Beauveria asiatica toxin; a Beauveria australis toxin; a Beauveria bassiana toxin; a Cordyceps bassiana toxin; a Beauveria brongniartii toxin; a Beauveria brumptii toxin; a Beauveria caledonica toxin; a Beauveria chiromensis toxin; a Beauveria coccorum toxin; a Beauveria cretacea toxin; a Beauveria cylindrospora toxin; a Beauveria delacroixii toxin; a Beauveria densa toxin; a Beauveria dependens tox
  • an IA can be a Beauveria bassiana toxin [00687] In some embodiments, an IA can be beauvericin. [00688] Beauvericin is a fungal toxin produced by various Fusarium species, as well as the fungus Beauveria bassiana. Beauvericin is a cyclic peptide, with toxic effects on insects as well as both human and murine cell lines. The activity of beauvericin is due to the ionophoric properties of the compound. Beauvericin is capable of forming complexes with alkali metal cations and affects ion transport across cell membranes.
  • an IA can be a beauvericin having the chemical formula C 45 H 57 N 3 O 9 .
  • an IA can be a “Beauvericin A” toxin having the chemical formula C 46 H 59 N 3 O 9 .
  • an IA can be a “Beauvericin B” toxin having the chemical formula C 47 H 61 N 3 O 9 .
  • an IA can be a Beauveria bassiana strain ANT-03 spore.
  • Exemplary methods of producing, making, and using fungi and fungal toxins for the control and/or inhibition of insects are disclosed in: U.S. Patent No.9,217,140, entitled “Fungal strain Beauveria sp. MTCC 5184 and a process for the preparation of enzymes therefrom”; U.S.
  • Lectins are polypeptides that are able to recognize and reversibly bind in a specific way to free carbohydrates and/or the glycoconjugates of cell membranes. Lectins are one of the two groups of glycan-binding proteins (GBPs), the other being sulfated glycosaminoglycan (GAG)-binding proteins. Found in the animalia, plantae, fungi, protista, archaea, bacteria and virus kingdoms, lectins have highly variable biological functions depending on the organism of origin.
  • lectins are involved in cell-extracellular matrix (ECM); gamete fertilization; cell-cell self-recognition; embryonic development; cell growth, differentiation, signaling, adhesion, and migration; apoptosis; host-pathogen interactions; immunomodulation and inflammation; glycoprotein folding and routing; mitogenic induction; and homeostasis.
  • ECM cell-extracellular matrix
  • lectins possess at least one non-catalytic domain with the ability to bind—in a reversible way with high specificity—to carbohydrates that are bound to cell membranes or free carbohydrates (e.g., polysaccharides, glycoproteins, or glycolipids). This domain is known as the carbohydrate-recognition domain (CRD).
  • CCD carbohydrate-recognition domain
  • examples of lectins can include: Concanavalin A (ConA), which is isolated from jack beans. ConA binds to glucose, mannose, and glycosides of mannose and/or glucose. Wheat germ agglutinin (WGA) is another lectin that binds to N-acetylglucosamine and its glycosides. Red kidney bean lectin binds to N-acetylglucosamine, and Peanut agglutinin binds to galactose and galactosides.
  • WGA Wheat germ agglutinin
  • Red kidney bean lectin binds to N-acetylglucosamine
  • Peanut agglutinin binds to galactose and galactosides.
  • lectins can be categorized according to several criteria, e.g., lectins can be categorized based on cell localization (e.g., extracellular lectins, intracellular endoplasmic reticulum (ER) lectins, Golgi lectins, cytoplasmic lectins, membrane-bound lectins). See Lakhtin et al., Lectins of living organisms. The overview. Anaerobe.2011 Dec;17(6):452-5, the disclosure of which is incorporated herein by reference in its entirety.
  • cell localization e.g., extracellular lectins, intracellular endoplasmic reticulum (ER) lectins, Golgi lectins, cytoplasmic lectins, membrane-bound lectins.
  • Similarities in structure or sequence can also be used to categorize lectins (e.g., beta prism lectins (B-type), calcium dependent lectins (C-type), lectins with Ficolins- Fibrinogen/collagen domain (F-type), garlic and snow drop lectins (G-type), hyaluronan bonding proteins or hyal-adherins (H-type), immunoglobulin superfamily lectins (I-type), jocob and related lectins (J-type), legume seed lectins (L-type), alpha mannosidase related lectins (M-type), nucleotide phosphohydrolases lectins (N-type), ricin lectins (R- type), Tachypleus tridentatus (T-type), wheat germ agglutinin (W type), Xenopus egg lectins (X type)).
  • lectins e.g., beta pris
  • carbohydrate specificities can also be used to categorize lectins.
  • animals and plants e.g., d-mannose (d-glucose)-binding lectins, 2- acetamido-2-deoxy-glucose-binding lectins, 2-acetamido-2-deoxy-galactose-binding lectins, d-galactose-binding lectins, l-fucose-binding lectins, other lectins
  • all organisms e.g., Glucose/mannose-binding lectins, galactose and N-acetyl-d-galactosamine- binding lectins, l-fucose-binding lectins, sialic acids-binding lectins.
  • Characterizing a lectin’s binding domain can be accomplished via X-ray co- crystallography, NMR, and MS mapping of relevant contacts and protein dynamics; equilibrium dialysis against labeled hapten; equilibrium binding with filtration (e.g., membranes); equilibrium binding, stopped by PEG with centrifugation (solubilized receptor); the use of multivalent ligands; the use of multivalent receptor probes; Biacore realtime kinetics; and/or evaluating the rates of cell adhesion, e.g., flow under shear to immobilized glycan or receptor.
  • an IA can be a lectin.
  • an IA can be a lectin, wherein said lectin is not fused nor operably linked to the CRIP.
  • an IA can be one of the following: Galanthus nivalis agglutinin (GNA); Sambucus nigra lectin (SNA); Maackia amurensis-II (MAL-II); Erythrina cristagalli lectin (ECL); Ricinus communis agglutinin-I (RCA); peanut agglutinin (PNA); wheat germ agglutinin (WGA); Griffonia simplicifolia-II (GSL-II); Con A; Lens culinaris agglutinin (LCA); Mannose-binding lectin (MBL); BanLec; galectins; Phaseolus vulgaris Leucoagglutinin (PHA-L); Phaseolus vulgaris Erythro
  • an IA can be one or more of the lectins listed in Table 3.
  • the lectins can have an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence set forth in any one of SEQ ID NOs
  • an IA may comprise an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% amino acid sequence identity to an amino acid sequence as set forth in any one of SEQ ID NOs: 35, 595-615.
  • IAs Chitinases
  • an IA may be a chitinase.
  • an IA may be a chitinase from Trichoderma viride.
  • an IA may be a chitinase having an amino acid sequence as set forth in SEQ ID NO: 620.
  • an IA may be a chitinase comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% amino acid sequence identity to an amino acid sequence as set forth in any one of SEQ ID NO: 620.
  • Azadirachta indica compounds [00714] Azadirachta indica (also known as neem, nimtree or Indian lilac) is a tree in the mahogany family, Meliaceae. Native to the Indian subcontinent, Azadirachta indica typically grows in tropical and semi-tropical regions. [00715] Azadirachta indica has been used for centuries as a source of pesticides.
  • neem seed extracts particularly the ones containing the hydrophilic, tetranortriterpenoid azadirachtin, are known to influence the feeding behavior, metamorphosis (insect growth regulating [IGR] effect), fecundity, and fitness of numerous insect species belonging to various orders.
  • Azadirachtin is a tetranortriterpenoid botanical insecticide of the liminoid class extracted from the neem tree (Azadirachta indica).
  • Azadirachtin is structurally similar to insect hormone “ecdysones”. These hormones typically control the process of metamorphosis when the insects pass from larva to pupa to adult. Azadirachtin mainly acts as an “ecdysone blocker”. It blocks the insect's production and release of vital hormones. As a result, insects cannot molt.
  • an IA can be an Azadirachta indica compound.
  • an IA can be an Azadirachtin; an Azadiradione; an Azadiradionolide; a Deacetylgedunin; a Deacetylazadirachtinol; a Desfuranoazadiradione; a Epoxyazadiradione; a Gedunin; a Mahmoodin; a Neemfruitin A; a Neemfruitin B; a Nimbolide; a Nimbin; a Nimolicinol; an Ohchinin Acetate; a Salannin; a Salannol; an alpha- Nimolactone; a beta-Nimolactone; a 2',3'-Dihydrosalannin; a 3-Deacetylsalannin; a 6- Deacetylnimbin; a 7-Acetyl-16,17-dehydro-16-hydroxyneotrichilenone; a 7-
  • an IA can be Azadirachtin.
  • an IA can be an Azadirachtin having a chemical formula: C 35 H 44 O 16 .
  • Exemplary methods of extracting Azadirachtin are disclosed in U.S. Patent No.6,312,738, entitled “Azadirachtin extraction process,” the disclosure of which is incorporated herein by reference in its entirety.
  • Exemplary methods of producing azadirachtin concentrates from neem seed materials are disclosed in PCT Application No.
  • WO1995002962A1 entitled “Method for producing azadirachtin concentrates from neem seed materials,” the disclosure of which is incorporated herein by reference in its entirety.
  • Exemplary methods of purifying azadirachtin are disclosed in U.S. Patent No. 5,736,145, entitled “Process for preparing purified Azadirachtin in powder form from neem seeds and storage stable aqueous composition containing Azadirachtin,” the disclosure of which is incorporated herein by reference in its entirety.
  • Exemplary methods of creating and storing compositions comprising azadirachtin are disclosed in U.S.
  • Patent No.6,811,790 entitled “Storage stable pesticide formulations containing azadirachtin,” the disclosure of which is incorporated herein by reference in its entirety.
  • Exemplary methods of making and using Azadirachtin are disclosed in U.S. Patent No.9,635,858, entitled “Pesticide and a method of controlling a wide variety of pests,” the disclosure of which is incorporated herein by reference in its entirety.
  • Exemplary Azadirachtin extracts and compositions are disclosed in U.S. Patent No.4,943,434, entitled “Insecticidal hydrogenated neem extracts”; U.S. Patent No.
  • an IA can be a boron compound.
  • an IA may be boric acid, diboron tetrahydroxide, a borate, a boron oxide, a borane, or any combination of any of the foregoing.
  • the IA may be a boranes and/or a borate ester that produces oxides of boron in aqueous media.
  • the boron compound is boric acid, a borate (e.g., basic sodium borate (borax)), or a mixture of boric acid and a borate.
  • an IA may be a borate. Suitable borates include, but are not limited to, perborates, metaborates, tetraborates, octaborates, borate esters, and any combination of any of the foregoing.
  • Preferred borates include, but are not limited to, metallic borates (e.g., sodium borate, zinc borate and potassium borate), such as disodium tetraborate decahydrate, disodium octaborate tetrahydrate, sodium metaborate, sodium perborate monohydrate, disodium octaborate, sodium tetraborate pentahydrate, sodium tetraborate, copper metaborate, zinc borate, barium metaborate, and any combination thereof.
  • metallic borates e.g., sodium borate, zinc borate and potassium borate
  • an IA can be borax (e.g., sodium borate decahydrate-10 mol Na 2 B 4 O 7 .10H 2 O or sodium borate pentahydrate-5 mol Na 2 B 4 O 7 .5H 2 O), [00735] In other embodiments, an IA can be a boron compound that may be utilized in effective amounts as substitutes for borax (or may be utilized in effective amounts in combination with borax or one another).
  • borax e.g., sodium borate decahydrate-10 mol Na 2 B 4 O 7 .10H 2 O or sodium borate pentahydrate-5 mol Na 2 B 4 O 7 .5H 2 O
  • an IA can be a boron compound that may be utilized in effective amounts as substitutes for borax (or may be utilized in effective amounts in combination with borax or one another).
  • an IA may be anhydrous borax (Na 2 B 4 O 7 ); ammonium tetraborate ((NH 4 ) 2 B 4 O 7 .4H 2 O); ammonium pentaborate ((NH 4 ) 2 B 10 O 16 .8H 2 O); potassium pentaborate (K 2 B 10 O 16 .8H 2 O); potassium tetraborate (K 2 B 4 O 7 .4H 2 O); sodium metaborate ((8 mol) Na 2 B 2 O 4 .8H 2 O); sodium metaborate ((4 mol) Na 2 B 2 O 4 .4H 2 O); disodium tetraborate decahydrate (Na 2 B 4 O 7 .10H 2 O); disodium tetraborate pentahydrate (Na 2 B 4 O 7 .5H 2 O); disodium octaborate tetrahydrate (Na 2 B 8 O 13 .4H 2 O); or combinations thereof.
  • anhydrous borax Na
  • an IA can be a boron compound that is selected from the group consisting of: borax, boric acid, disodium octaborate, sodium borate, sodium metaborate, sodium tetraborate decahydrate, boron oxide, boron carbide, boron nitride, boron tribromide, boron trichloride, and boron trifluoride.
  • an IA can be boric acid.
  • an IA can be boric acid having a chemical formula of H 3 BO 3 .
  • IAs Viruses
  • an IA can be a virus that possesses an insecticidal activity when in contact with an insect species.
  • an IA can be a DNA virus or an RNA virus.
  • an IA can be an ascovirus; baculovirus; densovirus; entomopoxvirus; hytrosavirus; iridovirus; nudivirus; polydnavirus; dicistrovirus; iflavirus; nodavirus; tetravirus; or cypovirus.
  • Ascoviridae family viruses [00745] In some embodiments, an IA can be a virus from the Ascoviridae family.
  • an IA can be an ascovirus such as Heliothis virescens ascovirus 3a; Heliothis virescens ascovirus 3; Heliothis virescens ascovirus 3b; Heliothis virescens ascovirus 3c; Heliothis virescens ascovirus 3d; Heliothis virescens ascovirus 3e; Heliothis virescens ascovirus 3f; Heliothis virescens ascovirus 3g; Heliothis virescens ascovirus 3h; Heliothis virescens ascovirus 3j; Spodoptera frugiperda ascovirus 1a; Trichoplusia ni ascovirus 2a; Heliothis virescens ascovirus 3i; Spodoptera ascovirus; Spodoptera exigua ascovirus 5a; Spodoptera frugiperda ascovirus 1c; Spodoptera frugiperda ascovirus 1d; Trichoplus
  • an IA can be a virus from the Ascoviridae family.
  • an IA can be a toursvirus such as Diadromus pulchellus toursvirus; Diadromus pulchellus ascovirus 4a; or Dasineura jujubifolia toursvirus 2a.
  • Densovirinae family viruses [00748] In some embodiments, an IA can be a virus from the Densovirinae family. For example, in some embodiments, an IA can be an Ambidensovirus.
  • an IA can be an Ambidensovirus selected from the following group: Asteroid ambidensovirus 1; Sea star-associated densovirus; Blattodean ambidensovirus 1; Periplaneta fuliginosa densovirus; Periplaneta fuliginosa densovirus Guo/2000; Blattodean ambidensovirus 2; Blattella germanica densovirus 1; Decapod ambidensovirus 1; Cherax quadricarinatus densovirus; Dipteran ambidensovirus 1; Culex pipiens densovirus; Hemipteran ambidensovirus 1; Planococcus citri densovirus; Hemipteran ambidensovirus 2; Dysaphis plantaginea densovirus; Hemipteran ambidensovirus 3; Myzus persicae densovirus; Myzus persicae nicotian
  • an IA can be a virus from the Entomopoxvirinae family.
  • an IA can be an Alphaentomopoxvirus; Betaentomopoxvirus; Diachasmimorpha entomopoxvirus; Melanoplus sanguinipes entomopoxvirus; or some heretofore unclassified Entomopoxvirinae.
  • an IA can be an Entomopoxvirinae family virus selected from the following group: Anomala cuprea entomopoxvirus; Adoxophyes honmai entomopoxvirus; Adoxophyes honmai entomopoxvirus 'L'; Amsacta moorei entomopoxvirus; Choristoneura biennis entomopoxvirus; Choristoneura fumiferana entomopoxvirus; Choristoneura rosaceana entomopoxvirus; Choristoneura rosaceana entomopoxvirus 'L'; Heliothis armigera entomopoxvirus; Mythimna separata entomopoxvirus; Mythimna separata entomopoxvirus 'L'; unclassified Betaentomopoxvirus; Diachas
  • an IA can be an Iridoviridae family virus, e.g., an Iridovirus.
  • an IA can be an Iridoviridae family virus selected from the following group: Tipula iridescent virus; Invertebrate iridescent virus 31; Armadillidium vulgare iridescent virus; Popillia japonica iridescent virus; Porcellio scaber iridescent virus; Invertebrate iridescent virus 6; Gryllus bimaculatus iridovirus; unclassified Iridovirus; Acetes erythraeus iridovirus; Anticarsia gemmatalis iridescent virus; Armadillidium decorum iridescent virus; Barramundi perch iridovirus; Bluegill sunfish iridovirus; Common ponyfish iridovirus; Crimson
  • an IA can be an Nudiviridae family virus, e.g., an Alphanudivirus, a Betanudivirus, or some heretofore unclassified Nudiviridae family virus [00758]
  • an IA can be an Nudiviridae family virus selected from the following group: Gryllus bimaculatus nudivirus; Oryctes rhinoceros nudivirus; Heliothis zea nudivirus; Helicoverpa zea nudivirus 2; Allomyrina virus; Drosophila innubila nudivirus; Drosophila nudivirus RLU-2011; Esparto virus; Homarus gammarus nudivirus; Kallithea virus; Macrobrachium nudivirus CN-SL2011; Mauternbach virus; Nilaparvata lugens endogenous nudivirus; Penaeus monodon n
  • an IA can be an Iflaviridae family virus selected from the following group: Antheraea pernyi iflavirus; Brevicoryne brassicae virus; Brevicoryne brassicae virus - UK; Deformed wing virus; Kakugo virus; VDV-1/DWV recombinant; Dinocampus coccinellae paralysis virus; Ectropis obliqua virus; Ectropis obliqua picorna-like virus; Infectious flacherie virus; Infectious flacherie virus isolate silkworm; Ixodes holocyclus iflavirus; Lygus lineolaris virus 1; Lymantria dispar iflavirus 1; Nilaparvata lugens honeydew virus 1; Perina nuda virus; Sacbrood virus; Sacbrood virus CSBV- LN/China/2009; Slow bee paralysis virus; Spodoptera exigua ifla
  • an IA can be a virus from the Baculoviridae family.
  • an IA can be an Alphabaculovirus, Betabaculovirus, Deltabaculovirus, Gammabaculovirus, or heretofore unclassified Baculoviridae virus.
  • an IA can be an Alphabaculovirus virus selected from the following group: Adoxophyes honmai nucleopolyhedrovirus; Agrotis ipsilon multiple nucleopolyhedrovirus; Agrotis segetum nucleopolyhedrovirus A; Agrotis segetum nucleopolyhedrovirus B; Antheraea pernyi nucleopolyhedrovirus; Antheraea proylei nucleopolyhedrovirus; Philosamia cynthia ricini nucleopolyhedrovirus virus; Anticarsia gemmatalis multiple nucleopolyhedrovirus; Autographa californica multiple nucleopolyhedrovirus; Anagrapha falcifera MNPV; Autographa californica nucleopolyhedrovirus; Galleria mellonella MNPV; Plutella xylostell
  • nucleopolyhedrovirus Hyphantria cunea nucleopolyhedrovirus; Lambdina fiscellaria nucleopolyhedrovirus; Leucania separata nucleopolyhedrovirus; Lonomia obliqua nucleopolyhedrovirus; Lonomia obliqua multiple nucleopolyhedrovirus; Lymantria dispar multiple nucleopolyhedrovirus; Lymantria xylina nucleopolyhedrovirus; Mamestra brassicae multiple nucleopolyhedrovirus; Mamestra configurata nucleopolyhedrovirus A; Mamestra configurata nucleopolyhedrovirus B; Helicoverpa armigera multiple nucleopolyhedrovirus; Maruca vitrata nucleopolyhedrovirus; Mythimna unipuncta nucleopolyhedrovirus; Operophtera
  • nucleopolyhedrovirus Malacosoma sp. alphabaculovirus; Malacosoma sp. nucleopolyhedrovirus; Neophasia sp. alphabaculovirus; or unidentified nuclear polyhedrosis viruses.
  • an IA can be a Betabaculovirus virus selected from the following group: Adoxophyes orana granulovirus; Agrotis segetum granulovirus; Artogeia rapae granulovirus; Pieris brassicae granulovirus; Choristoneura fumiferana granulovirus; Choristoneura occidentalis granulovirus; Clostera anachoreta granulovirus; Clostera anastomosis granulovirus A; Clostera anastomosis granulovirus Henan; Clostera anastomosis granulovirus B; Cnaphalocrocis medinalis granulovirus; Cryptophlebia leucotreta granulovirus; Cydia pomonella granulovirus; Cydia pomonella granulosis virus (isolate Mexican); Diatraea saccharalis granulovirus; Epinot
  • an IA can be a Deltabaculovirus virus selected from the following group: Culex nigripalpus nucleopolyhedrovirus; or Culex nigripalpus NPV Florida/1997.
  • an IA can be a Gammabaculovirus virus selected from the following group: Neodiprion lecontei nucleopolyhedrovirus; Neodiprion lecontei NPV (strain Canada); Neodiprion sertifer nucleopolyhedrovirus; unclassified Gammabaculovirus; or Neodiprion abietis NPV.
  • an IA can be some heretofore unclassified Baculoviridae virus selected from the following group: Achaea faber nucleopolyhedrovirus; Aedes disableans nucleopolyhedrovirus; Aglais urticae nucleopolyhedrovirus; Agraulis vanillae nucleopolyhedrovirus; Anomis sabulifera nucleopolyhedrovirus; Antheraea yamamai nucleopolyhedrovirus; Anthophila fabriciana granulovirus; Aroa discalis nucleopolyhedrovirus; Baculovirus penaei; Cadra cautella nucleopolyhedrovirus; Chaliopsis junodi nucleopolyhedrovirus; Cotesia marginiventris baculovirus; Cynosarga ornata nucleopolyhedrovirus; Darna
  • an IA can be a Baculoviridae virus [00769] In some embodiments, an IA can be a Betabaculovirus. [00770] In some embodiments, an IA can be a Adoxophyes orana granulovirus; a Agrotis segetum granulovirus; a Artogeia rapae granulovirus; a Pieris brassicae granulovirus; a Choristoneura fumiferana granulovirus; a Choristoneura occidentalis granulovirus; a Clostera anachoreta granulovirus; a Clostera anastomosis granulovirus A; a Clostera anastomosis granulovirus Henan; a Clostera anastomosis granulovirus B; a Cnaphalocrocis medinalis granulovirus; a Cryptophlebia leucotret
  • an IA can be a Cydia pomonella granulovirus.
  • an IA can be a Cydia pomonella granulovirus isolate V22 virus.
  • An exemplary complete genome of a Cydia pomonella granulovirus has NCBI Accession No. NC_002816.1; see also, Lugue et al., The complete sequence of the Cydia pomonella granulovirus genome. J Gen Virol.2001 Oct;82(Pt 10):2531-2547; the disclosures of which are incorporated herein by reference in their entireties.
  • IAs Bacteria and Bacterial Toxins
  • an IA can be a bacteria that possesses insecticidal activity when in contact with an insect.
  • an IA can be a peptide or toxin isolated from a bacteria.
  • an IA can be a bacterial toxin.
  • Photorhabdus and/or the toxins therefrom can be a bacterial toxin isolated from a bacteria belonging to the Xenorhabdus genus, or Photorhabdus genus.
  • an IA can be a Photorhabdus toxin.
  • an IA can be a Photorhabdus toxin selected from the group consisting of: Photorhabdus akhurstii toxin; Photorhabdus asymbiotica toxin; Photorhabdus asymbiotica subsp.
  • guanajuatensis toxin Photorhabdus uneii toxin; Photorhabdus laumondii toxin; Photorhabdus laumondii subsp. clarkei toxin; Photorhabdus laumondii subsp. laumondii toxin; Photorhabdus laumondii subsp. laumondii TTO1 toxin; Photorhabdus luminescens toxin; Photorhabdus luminescens BA1 toxin; Photorhabdus luminescens NBAII H75HRPL105 toxin; Photorhabdus luminescens NBAII HiPL101 toxin; Photorhabdus luminescens subsp.
  • luminescens toxin Photorhabdus luminescens subsp. luminescens ATCC 29999 toxin; Photorhabdus luminescens subsp. mexicana toxin; Photorhabdus luminescens subsp. sonorensis toxin; Photorhabdus namnaonensis toxin; Photorhabdus noenieputensis toxin; Photorhabdus stackebrandtii toxin; Photorhabdus tasmaniensis toxin; Photorhabdus temperata toxin; Photorhabdus temperata J3 toxin; Photorhabdus temperata subsp. phorame toxin; Photorhabdus temperata subsp.
  • an IA can be a Photorhabdus luminescens toxin.
  • an IA can be a Photorhabdus luminescens toxin, wherein the Photorhabdus luminescens toxin comprises a Photorhabdus luminescens “toxin complex a” (Tca).
  • an IA can be a Photorhabdus luminescens toxin, wherein the Photorhabdus luminescens toxin comprises a Photorhabdus luminescens “toxin complex c” (Tcc).
  • an IA can be a Photorhabdus luminescens toxin, wherein the Photorhabdus luminescens toxin comprises a Photorhabdus luminescens “toxin complex d” (Tcd).
  • an IA can be a Tca comprises a TcaA protein (SEQ ID NO: 616), a TcaB protein (SEQ ID NO: 617), a TcaC protein (SEQ ID NO: 618), and a TcaZ protein (SEQ ID NO: 619).
  • Patent No.6,528,484 entitled “Insecticidal protein toxins from Photorhabdus”; U.S. Patent No.7,777,100, entitled “DNA sequences from tcd genomic region of Photorhabdus luminescens”; U.S. Patent No. 7,161,062, entitled “DNA Sequences from Photorhabdus luminescens”; U.S. Patent Application Publication No.20030207806, entitled “Insecticidal protein toxins from Photorhabdus”; U.S. Patent Application Publication No.20070020625 A1, entitled “Sequence of the Photorhabdus luminescens strain tt01 genome and uses”; and U.S.
  • an IA can be one or more organisms belonging to the Yersinia genus.
  • an IA can be one or more peptides isolated from an organism belonging to the Yersinia genus.
  • an IA can be one or more of the following species: Yersinia aldovaeyb, Yersinia aleksiciae, Yersinia bercovieri, Yersinia canariae, Yersinia enterocolitica, Yersinia enterocolitica subsp. enterocolitica, Yersinia enterocolitica subsp. palearctica, Yersinia entomophaga, Yersinia frederiksenii, Yersinia hibernica, Yersinia intermedia, Yersinia kristensenii, Yersinia kristensenii subsp.
  • kristensenii Yersinia kristensenii subsp. rochesterensis, Yersinia massiliensis, Yersinia mollaretii, Yersinia nurmii, Yersinia pekkanenii, Yersinia pestis, Yersinia pestis subsp. pestis, Yersinia pestis subsp. medievalis, Yersinia pestis subsp. orientalis, Yersinia pseudotuberculosis, Yersinia pseudotuberculosis subsp. pestis, Yersinia pseudotuberculosis subsp. subsp.
  • an IA can be one or more peptides isolated from one or more of the following species: Yersinia aldovaeyb, Yersinia aleksiciae, Yersinia bercovieri, Yersinia canariae, Yersinia enterocolitica, Yersinia enterocolitica subsp. enterocolitica, Yersinia enterocolitica subsp.
  • an IA can be Yersinia entomophaga or Yersinia nurmii.
  • an IA can be one or more peptides isolated from Yersinia entomophaga or Yersinia nurmii.
  • Yersinia entomophaga is a gram-negative, rod-shaped, non-spore- forming bacterium isolated from diseased larvae of the New Zealand grass grub.
  • Yersinia nurmii is also a gram-negative, rod-shaped strain, albeit originating from broiler meat packaged under a modified atmosphere.
  • an IA can be a Yersinia entomophaga bacteria, and/or a toxin therefrom.
  • an IA can be one or more Yersinia nurmii bacteria, and/or a toxin therefrom. [00799] In some embodiments, an IA can be one or more Yersinia entomophaga bacteria and/or a toxin therefrom, and one or more Yersinia nurmii bacteria and/or a toxin therefrom. [00800] Exemplary methods of making and using Yersinia- and Yersinia-toxin- containing mixtures is disclosed in PCT Application No.
  • WO2018175677A1 entitled “COMBINATIONS OF YERSINIA ENTOMOPHAGA AND PESTICIDES OR OTHER SUBSTANCES” (Assignee: Novozymes Bioag A/S), the disclosure of which is incorporated herein by reference in its entirety.
  • Bacillus thuringiensis organisms, and the products therefrom Bacillus thuringiensis. The Bt bacteria produces a family of peptides that are toxic to many insects.
  • the Bt toxic peptides are well known for their ability to produce parasporal crystalline protein inclusions (usually referred to as crystals) that fall under two major classes of toxins; cytolysins (Cyt) and crystal Bt proteins (Cry). Since the cloning and sequencing of the first crystal proteins genes in the early-1980s, many others have been characterized and are now classified according to the nomenclature of Crickmore et al. (1998). Generally, Cyt proteins are toxic towards the insect orders Coleoptera (beetles) and Diptera (flies), and Cry proteins target Lepidopterans (moths and butterflies). Cry proteins bind to specific receptors on the membranes of mid-gut (epithelial) cells resulting in rupture of those cells.
  • Bt strains can have different complements of Cyt and Cry proteins, thus defining their host ranges. The genes encoding many Cry proteins have been identified.
  • holotype sequences assigns holotype sequences a unique name which incorporates ranks based on the degree of divergence, with the boundaries between the primary (Arabic numeral), secondary (uppercase letter), and tertiary (lower case letter) rank representing approximately 95%, 78% and 45% identities.
  • a fourth rank is used to indicate independent isolations of holotype toxin genes with sequences that are identical or differ only slightly.
  • crystalline or secreted pesticidal proteins such as the S-layer proteins (Pe ⁇ a et al., 2006) that are included here are, genetically altered crystal proteins, except those that were modified through single amino acid substitutions (e.g., Lambert et al., 1996). Any of these genes may be used to produce a suitable Bt related toxin for this invention.
  • Naturally occurring allelic variants can be identified with the use of well- known molecular biology techniques, such as polymerase chain reaction (PCR) and hybridization techniques as outlined below.
  • Variant nucleotide sequences also include synthetically derived nucleotide sequences that have been generated, for example, by using site-directed mutagenesis but which still encode the Bt protein proteins disclosed in the present disclosure as discussed below.
  • Variant proteins encompassed by the present disclosure are biologically active, that is they continue to possess the desired biological activity of the native protein, i.e., retaining pesticidal activity.
  • By “retains activity” is intended that the variant will have at least about 30%, at least about 50%, at least about 70%, or at least about 80% of the pesticidal activity of the native protein. Methods for measuring pesticidal activity are well known in the art. See, for example, Czapla and Lang (1990) J. Econ.
  • an IA can be a Bacillus thuringiensis organism.
  • an IA can be a Bacillus thuringiensis toxin.
  • an IA can be a Bacillus thuringiensis subspecies.
  • the Bacillus thuringiensis subspecies can be one of the following subspecies: aizawai; aizawai/pacificus; alesti; amagiensis; andalousiensis; argentinensis; asturiensis; azorensis; balearica; berliner; bolivia; brasilensis; cameroun; canadensis; chanpaisis; chinensis; colmeri; coreanensis; dakota; darmstadiensis; dendrolimus; entomocidus; entomocidus/subtoxicus; finitimus; fukuokaensis; galechiae; galleriae; graciosensis; guiyangiensis; higo; huazhongensis; iberica; indiana; israelensis; israelensis/tochigiensis; japonensis; jegathes
  • an IA can be a Bacillus thuringiensis var. or varietas.
  • an IA can be a Bacillus thuringiensis var. selected from the following group: Bacillus thuringiensis var. aizawai; Bacillus thuringiensis var. aizawai/pacificus; Bacillus thuringiensis var. alesti; Bacillus thuringiensis var. amagiensis; Bacillus thuringiensis var. andalousiensis; Bacillus thuringiensis var.
  • Bacillus thuringiensis var. asturiensis Bacillus thuringiensis var. azorensis; Bacillus thuringiensis var. balearica; Bacillus thuringiensis var. berliner; Bacillus thuringiensis var. bolivia; Bacillus thuringiensis var. brasilensis; Bacillus thuringiensis var. cameroun; Bacillus thuringiensis var. canadensis; Bacillus thuringiensis var. chanpaisis; Bacillus thuringiensis var. chinensis; Bacillus thuringiensis var.
  • Bacillus thuringiensis var. coreanensis Bacillus thuringiensis var. dakota; Bacillus thuringiensis var. darmstadiensis; Bacillus thuringiensis var. dendrolimus; Bacillus thuringiensis var. entomocidus; Bacillus thuringiensis var. entomocidus/subtoxicus; Bacillus thuringiensis var. finitimus; Bacillus thuringiensis var. fukuokaensis; Bacillus thuringiensis var. galechiae; Bacillus thuringiensis var. galleriae; Bacillus thuringiensis var.
  • Bacillus thuringiensis var. guiyangiensis Bacillus thuringiensis var. higo; Bacillus thuringiensis var. huazhongensis; Bacillus thuringiensis var. iberica; Bacillus thuringiensis var. indiana; Bacillus thuringiensis var. israelensis; Bacillus thuringiensis var. israelensis/tochigiensis; Bacillus thuringiensis var. japonensis; Bacillus thuringiensis var. jegathesan; Bacillus thuringiensis var.
  • Bacillus thuringiensis var. kenyae Bacillus thuringiensis var. kim
  • Bacillus thuringiensis var. kumamtoensis Bacillus thuringiensis var. kunthalanags3
  • Bacillus thuringiensis var. kunthalaRX24 Bacillus thuringiensis var. kunthalaRX27; Bacillus thuringiensis var. kunthalaRX28; Bacillus thuringiensis var. kurstaki; Bacillus thuringiensis var. kyushuensis; Bacillus thuringiensis var.
  • Bacillus thuringiensis var. londrina Bacillus thuringiensis var. malayensis; Bacillus thuringiensis var. medellin; Bacillus thuringiensis var. mexicanensis; Bacillus thuringiensis var. mogi; Bacillus thuringiensis var. monterrey; Bacillus thuringiensis var. morrisoni; Bacillus thuringiensis var. muju; Bacillus thuringiensis var. navarrensis; Bacillus thuringiensis var. neoleonensis; Bacillus thuringiensis var.
  • Bacillus thuringiensis var. novosibirsk Bacillus thuringiensis var. ostriniae
  • Bacillus thuringiensis var. oswaldocruzi Bacillus thuringiensis var. pahangi
  • Bacillus thuringiensis var. pakistani Bacillus thuringiensis var. palmanyolensis
  • Bacillus thuringiensis var. pirenaica Bacillus thuringiensis var. poloniensis
  • Bacillus thuringiensis var. pulsiensis Bacillus thuringiensis var. rongseni; Bacillus thuringiensis var. roskildiensis; Bacillus thuringiensis var. san diego; Bacillus thuringiensis var. seoulensis; Bacillus thuringiensis var. shandongiensis; Bacillus thuringiensis var. silo; Bacillus thuringiensis var. sinensis; Bacillus thuringiensis var. sooncheon; Bacillus thuringiensis var. sotto; Bacillus thuringiensis var.
  • Bacillus thuringiensis var. subtoxicus Bacillus thuringiensis var. sumiyoshiensis; Bacillus thuringiensis var. sylvestriensis; Bacillus thuringiensis var. tenebrionis; Bacillus thuringiensis var. thailandensis; Bacillus thuringiensis var. thompsoni; Bacillus thuringiensis var. thuringiensis; Bacillus thuringiensis var. tochigiensis; Bacillus thuringiensis var. toguchini; Bacillus thuringiensis var.
  • an IA can be a Bacillus thuringiensis serovar.
  • an IA can be a Bacillus thuringiensis serovar selected from the following group: Bacillus thuringiensis AKS-7; Bacillus thuringiensis Bt18247; Bacillus thuringiensis Bt18679; Bacillus thuringiensis Bt407; Bacillus thuringiensis DAR 81934; Bacillus thuringiensis DB27; Bacillus thuringiensis F14-1; Bacillus thuringiensis FC1; Bacillus thuringiensis FC10; Bacillus thuringiensis FC2; Bacillus thuringiensis FC6; Bacillus thuringiensis FC7; Bacillus thuringiensis FC8; Bacillus thuringiensis FC9; Bacillus thuringiensis HD-771; Bacillus thuringiensis HD-789; Bacillus thuringiensis HD1002; Bacillus thuringiensis IBL 200; Bacillus thuringiensis
  • an IA can be a one of the following organisms: Bacillus thuringiensis var. israelensis, Bacillus thuringiensis var. aizawai, Bacillus thuringiensis var. kurstaki, or Bacillus thuringiensis var. tenebrionensis.
  • an IA can be a protein isolated from Bacillus thuringiensis.
  • the IA can be a toxin isolated from Bacillus thuringiensis var. israelensis, Bacillus thuringiensis var. aizawai, Bacillus thuringiensis var. kurstaki, or Bacillus thuringiensis var. tenebrionensis.
  • an IA can be a MTX2 toxin, e.g., a MTX2 toxin isolated from Lysinibacillus sphaericus.
  • an IA can be a Bin-like toxin, e.g., a Bin-like toxin isolated from Lysinibacillus sphaericus.
  • an IA can be a Bacillus thuringiensis var. israelensis (Bti) toxin.
  • an IA can be a Bacillus thuringiensis ssp. israelensis Strain BMP 144 Bti toxin.
  • an IA can be a Bacillus thuringiensis var. kurstaki (Btk) toxin.
  • an IA can be a Bacillus thuringiensis ssp. kurstaki strain EVB-113-19 Btk toxin. [00825] In some embodiments, an IA can be a Bacillus thuringiensis var. tenebrionis (Btt) toxin. [00826] In some embodiments, an IA can be a Bacillus thuringiensis ssp. tenebrionis strain NB-176 Btt toxin. [00827] In some embodiments, the IA isolated from a Bacillus thuringiensis can be contained in a commercially available product.
  • the commercially available product comprising an IA can be AQUABAC XT® from Becker Microbial Products, Inc.; NOVODOR® FC from VALENT® U.S.A. LLC Agricultural Products; and/or BioProtec Plus TM from AEF Global Inc.
  • an IA can be one or more Bacillus thuringiensis ssp. kurstaki strain EVB-113-19 cells.
  • an IA can be one or more fermentation solids, spores, and/or insecticidal toxins isolated from Bacillus thuringiensis ssp. kurstaki strain EVB-113- 19 cells.
  • an IA can be one or more Bacillus thuringiensis ssp. tenebrionis strain NB-176 cells. [00831] In some embodiments, an IA can be one or more fermentation solids, spores, and/or insecticidal toxins isolated from Bacillus thuringiensis ssp. tenebrionis strain NB-176 cells. [00832] In some embodiments, an IA can be one or more Bacillus thuringiensis ssp. israelensis Strain BMP 144 cells.
  • an IA can be one or more fermentation solids, spores, and/or insecticidal toxins isolated from Bacillus thuringiensis ssp. israelensis Strain BMP 144 cells.
  • an IA can be AQUABAC XT®, consisting of the following ingredients: 6-10% ( ⁇ 8%) Bacillus thuringiensis ssp.
  • an IA can be NOVODOR® FC (or flowable concentrate), consisting of 10% Bacillus thuringiensis ssp.
  • an IA can be BioProtec Plus TM , consisting of 14.49% Bacillus thuringiensis ssp. kurstaki strain EVB-113-19 fermentation solids, spores, and insecticidal toxins with a potency of 17,500 Cabbage Looper Units (CLU) per mg of product (equivalent to 76 billion CLU per gallon of product); and 85.51% other/inactive ingredients.
  • CLU Cabbage Looper Units
  • an IA can be a Bt toxin, wherein said Bt toxin is a ⁇ - endotoxin (e.g., a Crystal (Cry) toxin and/or a cytolytic (Cyt) toxin); a vegetative insecticidal protein (Vip); a secreted insecticidal protein (Sip); or a Bin-like toxin.
  • a ⁇ - endotoxin e.g., a Crystal (Cry) toxin and/or a cytolytic (Cyt) toxin
  • Vip vegetative insecticidal protein
  • Sip secreted insecticidal protein
  • Bin-like toxin e.g., a Bin-like toxin.
  • an IA can be a Bt toxin having amino acid sequence as set forth in any one of SEQ ID NOs: 412-587.
  • an IA can be a Cry protein having amino acid sequence as set forth in any one of SEQ ID NOs: 412-461.
  • an IA can be a Cyt protein having amino acid sequence as set forth in any one of SEQ ID NOs: 462-481.
  • an IA can be a Vip having amino acid sequence as set forth in any one of SEQ ID NOs: 482-587.
  • an IA can be one or more of the following Cry proteins: Cry1Aa1, Cry1Aa2, Cry1Aa3, Cry1Aa4, Cry1Aa5, Cry1Aa6, Cry1Aa7, Cry1Aa8, Cry1Aa9, Cry1Aa10, Cry1Aa11, Cry1Aa12, Cry1Aa13, Cry1Aa14, Cry1Aa15, Cry1Aa16, Cry1Aa17, Cry1Aa18, Cry1Aa19, Cry1Aa20, Cry1Aa21, Cry1Aa22, Cry1Aa23, Cry1Aa24, Cry1Aa25, Cry1Ab1, Cry1Ab2, Cry1Ab3, Cry1Ab4, Cry1Ab5, Cry1Ab6, Cry1Ab7, Cry1Ab8, Cry1Ab9, Cry1Ab10, Cry1Ab11, Cry1Aa2, Cry1Aa3,
  • an IA can be any of the Cry toxins as described herein, or presented in Table 7. [00844] Table 7. Non-limiting examples of Cry toxins, their accession numbers on NCBI, and strain. Here, if a cell is left blank, then the accession number and/or strain is not applicable.
  • an IA can be one or more of the following Cyt proteins: Cyt1Aa1, Cyt1Aa2, Cyt1Aa3, Cyt1Aa4, Cyt1Aa5, Cyt1Aa6, Cyt1Aa7, Cyt1Aa8, Cyt1Aa-like, Cyt1Ab1, Cyt1Ba1, Cyt1Ca1, Cyt1Da1, Cyt1Da2, Cyt2Aa1, Cyt2Aa2, Cyt2Aa3, Cyt2Aa4, Cyt2Ba1, Cyt2Ba2, Cyt2Ba3, Cyt2Ba4, Cyt2Ba5, Cyt2Ba6, Cyt2Ba7, Cyt2Ba8, Cyt2Ba9, Cyt2Ba10, Cyt2Ba11, Cyt2Ba12, Cyt2Ba13, Cyt2Ba14, Cyt2Ba15, Cyt2Ba16, Cyt2Ba-like, Cyt2Bb1, Cyt1Aa1, Cyt1A
  • an IA can be any Cyt toxin as described herein, or presented in Table 8. [00847] Table 8. Non-limiting examples of Cyt toxins, their accession numbers on NCBI, and strain. Here, if a cell is left blank, then the accession number and/or strain is not applicable. [00848] In some embodiments, an IA can be a protein belonging to the Vip1, Vip2, Vip3, or Vip4 family.
  • an IA can be one or more of the following Vip proteins: Vip1Aa1, Vip1Aa2, Vip1Aa3, Vip1Ab1, Vip1Ac1, Vip1Ad1, Vip1Ba1, Vip1Ba2, Vip1Bb1, Vip1Bb2, Vip1Bb3, Vip1Bc1, Vip1Ca1, Vip1Ca2, Vip1Da1, Vip2Aa1, Vip2Aa2, Vip2Aa3, Vip2Ab1, Vip2Ac1, Vip2Ac2, Vip2Ad1, Vip2Ae1, Vip2Ae2, Vip2Ae3, Vip2Af1, Vip2Af2, Vip2Ag1, Vip2Ag2, Vip2Ba1, Vip2Ba2, Vip2Bb1, Vip2Bb2, Vip2Bb3, Vip2Bb4, Vip3Aa1, Vip3Aa2, Vip3Aa3, Vip3Aa4,
  • an IA can be any Vip protein as described herein, or presented in Table 9.
  • Table 9 Non-limiting examples of Vip proteins and their accession numbers on NCBI. Here, if a cell is left blank, then the accession number is not applicable.
  • CRIP AND IA COMBINATIONS Any of the aforementioned CRIPs or IAs can be used to create a mixture and/or composition of the present invention, wherein said mixture and/or composition comprises at least one CRIP, and at least one other IA.
  • Described and incorporated by reference to the polypeptides identified herein are homologous variants of sequences mentioned, having homology to such sequences or referred to herein, including all homologous sequences having at least any of the following percent identities to any of the sequences disclosed here or to any sequence incorporated by reference: 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or greater identity, or 100% identity to any and all sequences identified in the sequences noted above, and to any other sequence identified herein, including each and every sequence in the sequence listing of this application.
  • homologous or homology when used herein with a number such as 50% or greater, then what is meant is percent identity or percent similarity between the two peptides. When homologous or homology is used without a numeric percent then it refers to two peptide sequences that are closely related in the evolutionary or developmental aspect in that they share common physical and functional aspects, like topical toxicity and similar size (i.e., the homolog being within 100% greater length or 50% shorter length of the peptide specifically mentioned herein or identified by reference herein as above). [00854] In some embodiments a mixture can consist of any CRIP described herein, and any IA described herein.
  • a mixture can comprise one or more CRIPs combined with one or more IAs; and wherein the mixture further comprises an excipient.
  • CRIP and IA Combinations [00857] In some embodiments, any one or more of the CRIPs presented in Table A may be combined with any one or more of the IAs presented in Table B.
  • Table A and Table B below presents preferred CRIPs and IAs of the present invention, respectfully. Both tables provide a “Group No.” and a “Group Name”: these designations identify the category a given CRIP or IA belongs to. CRIP Group Nos are preceded by the Roman numeral “I” (e.g., I1, I2, I3, etc.).
  • IA Insecticidal Agent
  • Group Nos are preceded by the Roman numeral “II” (e.g., II2, II3, II3, etc.).
  • CRIP No.” and IA No.” these designations identify specific CRIPs or IAs.
  • the CRIP Nos are preceded by an “A” (e.g., A1, A2, A3, etc.).
  • IA Nos are preceded by a “B” (e.g., B1, B2, B3, etc.).
  • Table A CRIPs of the present invention.
  • Table B Insecticidal Agents (IAs) of the present invention.
  • the asterisk “*” indicates an IA that was shown to not exert a greater than additive insecticidal effect when combined with U+2-ACTX-Hv1a (SEQ ID NO: 61).
  • 118 119 120 121 122 123 124 125 126 1 27 128 1 29 130 1 31 132 133 134 135 136 137 138 139 140 141 142 143 144 1 45 146 1 47 148 218 219 220 221 222 223 224 225 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290
  • a mixture of the present invention may comprise, consist essentially of, or consist of, one or more CRIPs selected from any one of CRIP Groups I1; I2; I3; I4; I5; I6; I7, or combination thereof; combined with any one or more IAs in IA Groups II1; II2; II3; II4; II5; II6; II7; II8; II9; II10; II11; II12; II13; II14; II15; II16; II17; II18; II19; II20; II21; II22; II23; II24; II25; II26; II27; II28; II29; II30; II31; II32; II33; II34; II35; II36; II37; II38; II39; II40; II41; II42; II43; II44; II45, or combination thereof.
  • a mixture of the present invention may comprise, consist essentially of, or consist of, a CRIP selected from: A1; A2; A3; A4; A5; A6; A7; A8; A9; A10; A11; A12; A13; A14; A15; A16; A17; A18; A19; A20; A21; A22; A23; A24; A25; A26; A27; A28; A29; A30; A31; A32; A33; A34; A35; A36; A37; A38; A39; A40; A41; A42; A43; A44; A45; A46; A47; A48; A49; A50; A51; A52; A53; A54; A55; A56; A57; A58; A59; A60; A61; A62; A63; A64; A65; A66; A67; A68; or a combination thereof; combined with an IA selected from: B
  • a mixture of the present invention may comprise, consist essentially of, or consist of, the CRIP A1 from Table A, combined with any one or more Insecticidal Agents (IAs) selected from the group consisting of B1-B479, in Table B.
  • IAs Insecticidal Agents
  • a mixture of the present invention may comprise, consist essentially of, or consist of, the CRIP A2 from Table A, combined with any one or more Insecticidal Agents (IAs) selected from the group consisting of B1-B479, in Table B.
  • a mixture of the present invention may comprise, consist essentially of, or consist of, the CRIP A3 from Table A, combined with any one or more Insecticidal Agents (IAs) selected from the group consisting of B1-B479, in Table B.
  • IAs Insecticidal Agents
  • a mixture of the present invention may comprise, consist essentially of, or consist of, the CRIP A4 from Table A, combined with any one or more Insecticidal Agents (IAs) selected from the group consisting of B1-B479, in Table B.
  • a mixture of the present invention may comprise, consist essentially of, or consist of, the CRIP A5 from Table A, combined with any one or more Insecticidal Agents (IAs) selected from the group consisting of B1-B479, in Table B.
  • IAs Insecticidal Agents
  • a mixture of the present invention may comprise, consist essentially of, or consist of, the CRIP A6 from Table A, combined with any one or more Insecticidal Agents (IAs) selected from the group consisting of B1-B479, in Table B.
  • a mixture of the present invention may comprise, consist essentially of, or consist of, the CRIP A7 from Table A, combined with any one or more Insecticidal Agents (IAs) selected from the group consisting of B1-B479, in Table B.
  • IAs Insecticidal Agents
  • a mixture of the present invention may comprise, consist essentially of, or consist of, the CRIP A8 from Table A, combined with any one or more Insecticidal Agents (IAs) selected from the group consisting of B1-B479, in Table B.
  • a mixture of the present invention may comprise, consist essentially of, or consist of, the CRIP A9 from Table A, combined with any one or more Insecticidal Agents (IAs) selected from the group consisting of B1-B479, in Table B.
  • IAs Insecticidal Agents
  • a mixture of the present invention may comprise, consist essentially of, or consist of, the CRIP A10 from Table A, combined with any one or more Insecticidal Agents (IAs) selected from the group consisting of B1-B479, in Table B.
  • a mixture of the present invention may comprise, consist essentially of, or consist of, the CRIP A11 from Table A, combined with any one or more Insecticidal Agents (IAs) selected from the group consisting of B1-B479, in Table B.
  • IAs Insecticidal Agents
  • a mixture of the present invention may comprise, consist essentially of, or consist of, the CRIP A12 from Table A, combined with any one or more Insecticidal Agents (IAs) selected from the group consisting of B1-B479, in Table B.
  • a mixture of the present invention may comprise, consist essentially of, or consist of, the CRIP A13 from Table A, combined with any one or more Insecticidal Agents (IAs) selected from the group consisting of B1-B479, in Table B.
  • IAs Insecticidal Agents
  • a mixture of the present invention may comprise, consist essentially of, or consist of, the CRIP A14 from Table A, combined with any one or more Insecticidal Agents (IAs) selected from the group consisting of B1-B479, in Table B.
  • a mixture of the present invention may comprise, consist essentially of, or consist of, the CRIP A15 from Table A, combined with any one or more Insecticidal Agents (IAs) selected from the group consisting of B1-B479, in Table B.
  • IAs Insecticidal Agents
  • a mixture of the present invention may comprise, consist essentially of, or consist of, the CRIP A16 from Table A, combined with any one or more Insecticidal Agents (IAs) selected from the group consisting of B1-B479, in Table B.
  • a mixture of the present invention may comprise, consist essentially of, or consist of, the CRIP A17 from Table A, combined with any one or more Insecticidal Agents (IAs) selected from the group consisting of B1-B479, in Table B.
  • IAs Insecticidal Agents
  • a mixture of the present invention may comprise, consist essentially of, or consist of, the CRIP A18 from Table A, combined with any one or more Insecticidal Agents (IAs) selected from the group consisting of B1-B479, in Table B.
  • a mixture of the present invention may comprise, consist essentially of, or consist of, the CRIP A19 from Table A, combined with any one or more Insecticidal Agents (IAs) selected from the group consisting of B1-B479, in Table B.
  • IAs Insecticidal Agents
  • a mixture of the present invention may comprise, consist essentially of, or consist of, the CRIP A20 from Table A, combined with any one or more Insecticidal Agents (IAs) selected from the group consisting of B1-B479, in Table B.
  • a mixture of the present invention may comprise, consist essentially of, or consist of, the CRIP A21 from Table A, combined with any one or more Insecticidal Agents (IAs) selected from the group consisting of B1-B479, in Table B.
  • IAs Insecticidal Agents
  • a mixture of the present invention may comprise, consist essentially of, or consist of, the CRIP A22 from Table A, combined with any one or more Insecticidal Agents (IAs) selected from the group consisting of B1-B479, in Table B.
  • a mixture of the present invention may comprise, consist essentially of, or consist of, the CRIP A23 from Table A, combined with any one or more Insecticidal Agents (IAs) selected from the group consisting of B1-B479, in Table B.
  • IAs Insecticidal Agents
  • a mixture of the present invention may comprise, consist essentially of, or consist of, the CRIP A24 from Table A, combined with any one or more Insecticidal Agents (IAs) selected from the group consisting of B1-B479, in Table B.
  • a mixture of the present invention may comprise, consist essentially of, or consist of, the CRIP A25 from Table A, combined with any one or more Insecticidal Agents (IAs) selected from the group consisting of B1-B479, in Table B.
  • IAs Insecticidal Agents
  • a mixture of the present invention may comprise, consist essentially of, or consist of, the CRIP A26 from Table A, combined with any one or more Insecticidal Agents (IAs) selected from the group consisting of B1-B479, in Table B.
  • a mixture of the present invention may comprise, consist essentially of, or consist of, the CRIP A27 from Table A, combined with any one or more Insecticidal Agents (IAs) selected from the group consisting of B1-B479, in Table B.
  • IAs Insecticidal Agents
  • a mixture of the present invention may comprise, consist essentially of, or consist of, the CRIP A28 from Table A, combined with any one or more Insecticidal Agents (IAs) selected from the group consisting of B1-B479, in Table B.
  • a mixture of the present invention may comprise, consist essentially of, or consist of, the CRIP A29 from Table A, combined with any one or more Insecticidal Agents (IAs) selected from the group consisting of B1-B479, in Table B.
  • IAs Insecticidal Agents
  • a mixture of the present invention may comprise, consist essentially of, or consist of, the CRIP A30 from Table A, combined with any one or more Insecticidal Agents (IAs) selected from the group consisting of B1-B479, in Table B.
  • a mixture of the present invention may comprise, consist essentially of, or consist of, the CRIP A31 from Table A, combined with any one or more Insecticidal Agents (IAs) selected from the group consisting of B1-B479, in Table B.
  • IAs Insecticidal Agents
  • a mixture of the present invention may comprise, consist essentially of, or consist of, the CRIP A32 from Table A, combined with any one or more Insecticidal Agents (IAs) selected from the group consisting of B1-B479, in Table B.
  • a mixture of the present invention may comprise, consist essentially of, or consist of, the CRIP A33 from Table A, combined with any one or more Insecticidal Agents (IAs) selected from the group consisting of B1-B479, in Table B.
  • IAs Insecticidal Agents
  • a mixture of the present invention may comprise, consist essentially of, or consist of, the CRIP A34 from Table A, combined with any one or more Insecticidal Agents (IAs) selected from the group consisting of B1-B479, in Table B.
  • a mixture of the present invention may comprise, consist essentially of, or consist of, a CRIP from Table A selected from: A1; A2; A3; A4; A5; A6; A7; A8; A9; A10; A11; A12; A13; A14; A15; A16; A17; A18; A19; A20; A21; A22; A23; A24; A25; A26; A27; A28; A29; A30; A31; A32; A33; A34; A35; A36; A37; A38; A39; A40; A41; A42; A43; A44; A45; A46; A47; A48; A49; A50; A51; A52; A53; A54; A55; A56; A57; A58; A59; A60; A61; A62; A63; A64; A65; A66; A67; or A68; wherein said CRIP is at least 50% identical,
  • a mixture of the present invention may comprise, consist essentially of, or consist of, a CRIP and an IA; wherein the CRIP peptide may comprise, consist essentially of, or consist of, an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% amino acid sequence identity to: a spider peptide having an amino acid sequence as set forth in any one of SEQ ID NOs: 192-370
  • the ratio of CRIP to IA, on a dry weight basis can be selected from at least about the following ratios: 10,000:1, 5,000:1, 1,000:1, 500:1, 250:1, 200:1, 100:1, 99:1, 95:5, 90:10, 85:15, 80:20, 75:25, 70:30, 65:35, 60:40, 55:45, 1:1, 45:55, 40:60, 35:65, 30:70, 25:75, 20:80, 15:85, 10:90, 5:95, 1:99, 1:100, 1:200, 1:250, 1:500, 1:1,000, 1:5,000, or 1:10,000, or any combination of any two of these values.
  • the total concentration of CRIP and IA in the mixture is selected from the following percent concentrations: 0, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99 or 100%, or any range between any two of these values, and the remaining percentage of the mixture is comprised of excipients.
  • Any of the foregoing mixtures, compositions, or formulations comprising one or more CRIPs and one or more IAs as described herein, can further comprise, consist essentially of, or consist of, an excipient.
  • any of the foregoing mixtures or compositions comprising one or more CRIPs and one or more IAs can be applied concomitantly and/or sequentially, and either in the same or separate compositions.
  • the ratios of CRIP to IA will depend on the insect pest to be targeted, and the needs of the user.
  • any of the foregoing mixtures or compositions comprising one or more CRIPs and one or more IAs can be can be applied to the crop area or plant to be treated, simultaneously or in succession, with other compounds.
  • these other compounds can be fertilizers, weed killers, cryoprotectants, surfactants, detergents, pesticidal soaps, dormant oils, polymers, and/or time-release or biodegradable carrier formulations that permit long-term dosing of a target area following a single application of the formulation.
  • the other compounds can also be selective herbicides, chemical insecticides, virucides, microbicides, amoebicides, pesticides, fungicides, bacteriocides, nematocides, molluscicides or mixtures of several of these preparations, if desired, together with further agriculturally acceptable carriers, surfactants or application- promoting adjuvants customarily employed in the art of formulation.
  • suitable carriers and adjuvants can be solid or liquid, and correspond to the substances ordinarily employed in formulation technology, e.g. natural or regenerated mineral substances, solvents, dispersants, wetting agents, tackifiers, binders or fertilizers.
  • any of the foregoing mixtures, compositions, or formulations may be prepared into edible “baits” or fashioned into pest “traps” to permit feeding or ingestion by a target pest of the pesticidal formulation.
  • v/v or “% v/v” or “volume per volume” refers to the volume concentration of a solution (“v/v” stands for volume per volume).
  • v/v can be used when both components of a solution are liquids.
  • w/w or “% w/w” or “weight per weight” or “wt/wt” or “% wt/wt” refers to the weight concentration of a formulation or solution, i.e., percent weight in weight (“w/w” stands for weight per weight).
  • w/w expresses the number of grams (g) of a constituent in 100 g of solution or mixture.
  • a mixture consisting of 30 g of ingredient X, and 70 g of water would be expressed as “ingredient X 30% w/w.”
  • Percent weight per weight (% w/w) is calculated as follows: (weight of solute (g)/ weight of solution (g)) x 100; or (mass of solute (g)/ mass of solution (g)) x 100.
  • w/v or “% w/v” or “weight per volume” refers to the mass concentration of a solution, i.e., percent weight in volume (“w/v” stands for weight per volume).
  • w/v expresses the number of grams (g) of a constituent in 100 mL of solution.
  • compositions comprising, consisting essentially of, or consisting of a (1) CRIP, a CRIP-insecticidal protein, a pharmaceutically acceptable salt thereof, or a combination thereof; and (2) one or more Insecticidal Agents, for example, agrochemical compositions, can include, but are not limited to, aerosols and/or aerosolized products, e.g., sprays, fumigants, powders, dusts, and/or gases; seed dressings; oral preparations (e.g., insect food, etc.); transgenic organisms expressing and/or producing a TVP, a TVP-insecticidal protein, and/or a TVP ORF (either transiently and/or stably), e.g., a plant or an animal.
  • aerosols and/or aerosolized products e.g., sprays, fumigants, powders, dusts, and/or gases
  • seed dressings e.g., oral preparations (e.g.
  • the composition may be formulated as a powder, dust, pellet, granule, spray, emulsion, colloid, solution, or such like, and may be prepared by such conventional means as desiccation, lyophilization, homogenization, extraction, filtration, centrifugation, sedimentation, or concentration of a culture of cells comprising the polypeptide.
  • the polypeptide may be present in a concentration of from about 1% to about 99% by weight.
  • the pesticide compositions described herein may be made by formulating either the CRIP, CRIP-insecticidal protein, or pharmaceutically acceptable salt thereof, with the desired agriculturally-acceptable carrier.
  • compositions may be formulated prior to administration in an appropriate means such as lyophilized, freeze-dried, desiccated, or in an aqueous carrier, medium or suitable diluent, such as saline and/or other buffer.
  • the formulated compositions may be in the form of a dust or granular material, or a suspension in oil (vegetable or mineral), or water or oil/water emulsions, or as a wettable powder, or in combination with any other carrier material suitable for agricultural application.
  • Suitable agricultural carriers can be solid or liquid and are well known in the art.
  • the formulations may be mixed with one or more solid or liquid adjuvants and prepared by various means, e.g., by homogeneously mixing, blending and/or grinding the pesticidal composition with suitable adjuvants using conventional formulation techniques. Suitable formulations and application methods are described in U.S. Pat. No.6,468,523, the disclosure of which is incorporated by reference herein in its entirety.
  • the composition comprises, consists essentially of, or consists of: a CRIP, or pharmaceutically acceptable salt thereof; an Insecticidal Agent; and an excipient.
  • the composition comprises, consists essentially of, or consists of: a CRIP-insecticidal protein, or pharmaceutically acceptable salt thereof; an Insecticidal Agent; and an excipient.
  • the composition comprises, consists essentially of, or consists of: (1) a CRIP, or pharmaceutically acceptable salt thereof; a CRIP-insecticidal protein, or pharmaceutically acceptable salt thereof; or a combination thereof; (2) one or more Insecticidal Agents; and (3) an excipient.
  • Pharmaceutically acceptable salts [00917] As used herein, the term “pharmaceutically acceptable salt” and “agriculturally acceptable salt” are synonymous.
  • a pharmaceutically acceptable salt of the present invention possesses the desired pharmacological activity of the parent compound.
  • Such salts include: acid addition salts, formed with inorganic acids; acid addition salts formed with organic acids; or salts formed when an acidic proton present in the parent compound is replaced by a metal ion, e.g., an alkali metal ion, aluminum ion; or coordinates with an organic base such as ethanolamine, and the like.
  • pharmaceutically acceptable salts include conventional toxic or non-toxic salts.
  • convention non-toxic salts include those such as fumarate, phosphate, citrate, chlorydrate, and the like.
  • the pharmaceutically acceptable salts of the present invention can be synthesized from a parent compound by conventional chemical methods.
  • such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two.
  • non-aqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Lists of suitable salts are found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p.1418, the disclosure of which is incorporated herein by reference in its entirety.
  • a pharmaceutically acceptable salt can be one of the following: hydrochloride; sodium; sulfate; acetate; phosphate or diphosphate; chloride; potassium; maleate; calcium; citrate; mesylate; nitrate; tartrate; aluminum; or gluconate.
  • a list of pharmaceutically acceptable acids that can be used to form salts can be: glycolic acid; hippuric acid; hydrobromic acid; hydrochloric acid; isobutyric acid; lactic acid (DL); lactobionic acid; lauric acid; maleic acid; malic acid (- L); malonic acid; mandelic acid (DL); methanesulfonic acid ; naphthalene-1,5-disulfonic acid; naphthalene-2-sulfonic acid; nicotinic acid; nitric acid; oleic acid; oxalic acid; palmitic acid; pamoic acid; phosphoric acid; proprionic acid; pyroglutamic acid (- L); salicylic acid; sebacic acid; stearic acid; succinic acid; sulfuric acid; tartaric acid (+ L); thiocyanic acid; toluenesulfonic acid (p); undecylenic acid; a
  • pharmaceutically acceptable salt can be any organic or inorganic addition salt.
  • the salt may use an inorganic acid and an organic acid as a free acid.
  • the inorganic acid may be hydrochloric acid, bromic acid, nitric acid, sulfuric acid, perchloric acid, phosphoric acid, etc.
  • the organic acid may be citric acid, acetic acid, lactic acid, maleic acid, fumaric acid, gluconic acid, methane sulfonic acid, gluconic acid, succinic acid, tartaric acid, galacturonic acid, embonic acid, glutamic acid, aspartic acid, oxalic acid, (D) or (L) malic acid, maleic acid, methane sulfonic acid, ethane sulfonic acid, 4- toluene sulfonic acid, salicylic acid, citric acid, benzoic acid, malonic acid, etc.
  • the salts include alkali metal salts (sodium salts, potassium salts, etc.) and alkaline earth metal salts (calcium salts, magnesium salts, etc.).
  • the acid addition salt may include acetate, aspartate, benzoate, besylate, bicarbonate/carbonate, bisulfate/sulfate, borate, camsylate, citrate, edisilate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hibenzate, hydrochloride/chloride, hydrobromide/bromide, hydroiodide/iodide, isethionate, lactate, malate, maleate, malonate, mesylate, methyl sulfate, naphthalate, 2-napsylate, nicotinate, nitrate, orotate, oxalate, palmitate,
  • the pharmaceutically acceptable salt can be a salt with an acid such as acetic acid, propionic acid, butyric acid, formic acid, trifluoroacetic acid, maleic acid, tartaric acid, citric acid, stearic acid, succinic acid, ethylsuccinic acid, lactobionic acid, gluconic acid, glucoheptonic acid, benzoic acid, methanesulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, laurylsulfuric acid, malic acid, aspartic acid, glutaminic acid, adipic acid, cysteine, N- acetylcysteine, hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, hydroiodic acid, nicotinic acid, oxalic acid, picric acid
  • an acid such as acetic
  • the pharmaceutically acceptable salt can be prepared from either inorganic or organic bases.
  • Salts derived from inorganic bases include, but are not limited to, the sodium, potassium, lithium, ammonium, calcium, magnesium, ferrous, zinc, copper, manganous, aluminum, ferric, manganic salts, and the like.
  • Preferred inorganic salts are the ammonium, sodium, potassium, calcium, and magnesium salts.
  • Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally-occurring substituted amines, and cyclic amines, including isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol, tromethamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, N-alkylglucamines, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, and the like.
  • Preferred organic bases are isopropylamine, diethylamine, ethanolamine, piperidine, tromethamine, and choline.
  • pharmaceutically acceptable salt refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio.
  • Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge, et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 66: 1–19 (1977), the disclosure of which is incorporated herein by reference in its entirety.
  • the salts of the present invention can be prepared in situ during the final isolation and purification of the compounds of the invention, or separately by reacting the free base function with a suitable organic acid.
  • suitable organic acid examples include salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange.
  • salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2- naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate
  • Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like.
  • Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate and aryl sulfonate.
  • Exemplary descriptions of pharmaceutically acceptable salts is provided in P. H. Stahl and C. G. Wermuth, (editors), Handbook of Pharmaceutical Salts: Properties, Selection and Use, John Wiley & Sons, Aug 23, (2002), the disclosure of which is incorporated herein by reference in its entirety.
  • Sprayable Compositions can include field sprayable formulations for agricultural usage and indoor sprays for use in interior spaces in a residential or commercial space.
  • residual sprays or space sprays comprising a combination of one or more of CRIPs: A1-A68, and one or more Insecticidal Agents: B1-B479, can be used to reduce or eliminate insect pests in an interior space.
  • Surface spraying indoors is the technique of applying a variable volume sprayable volume of an insecticide onto indoor surfaces where vectors rest, such as on walls, windows, floors and ceilings.
  • variable volume sprayable volume is to reduce the lifespan of the insect pest, (for example, a fly, a flea, a tick, or a mosquito vector) and thereby reduce or interrupt disease transmission.
  • the secondary impact is to reduce the density of insect pests within the treatment area.
  • SSI can be used as a method for the control of insect pest vector diseases, such as Lyme disease, Salmonella, Chikungunya virus, Zika virus, and malaria, and can also be used in the management of parasites carried by insect vectors, such as Leishmaniasis and Chagas disease.
  • Many mosquito vectors that harbor Zika virus, Chikungunya virus, and malaria include endophilic mosquito vectors, resting inside houses after taking a blood meal.
  • SSI surface spraying indoors
  • a sprayable composition comprising a combination of (1) one or more CRIPs set for the in Table A, i.e., A1-A68, (2) one or more Insecticidal Agents set forth in Table B, i.e., B1-B479, and (3) an excipient.
  • SSI involves applying the composition onto the walls and other surfaces of a house with a residual insecticide.
  • the composition comprising a combination of (1) one or more CRIPs set for the in Table A, i.e., A1-A68, (2) one or more Insecticidal Agents set forth in Table B, i.e., B1-B479, and (3) an excipient, will knock down insect pests that come in contact with these surfaces.
  • SSI does not directly prevent people from being bitten by mosquitoes. Rather, it usually controls insect pests after they have blood fed, if they come to rest on the sprayed surface. SSI thus prevents transmission of infection to other persons. To be effective, SSI must be applied to a very high proportion of households in an area (usually greater than 40-80 percent).
  • sprays in accordance with the invention having good residual efficacy and acceptable odor are particularly suited as a component of integrated insect pest vector management or control solutions.
  • SSI which requires that the active CRIP, CRIP-insecticidal protein, or Insecticidal Agent be bound to surfaces of dwellings, such as walls or ceilings, as with a paint, for example, space spray products of the invention rely on the production of a large number of small insecticidal droplets intended to be distributed through a volume of air over a given period of time.
  • the traditional methods for generating a space-spray include thermal fogging (whereby a dense cloud of a composition comprising a combination of (1) one or more CRIPs set for the in Table A, i.e., A1-A68, (2) one or more Insecticidal Agents set forth in Table B, i.e., B1-B479, and (3) an excipient, is produced giving the appearance of a thick fog) and Ultra Low Volume (ULV), whereby droplets are produced by a cold, mechanical aerosol-generating machine.
  • thermal fogging whereby a dense cloud of a composition comprising a combination of (1) one or more CRIPs set for the in Table A, i.e., A1-A68, (2) one or more Insecticidal Agents set forth in Table B, i.e., B1-B479, and (3) an excipient, is produced giving the appearance of a thick fog
  • UUV Ultra Low Volume
  • Ready-to-use aerosols such as aerosol cans may also be used.
  • the foregoing method is a very effective way to rapidly reduce the population of flying insect pests in a specific area. And, because there is very limited residual activity from the application, it must be repeated at intervals of 5-7 days in order to be fully effective. This method can be particularly effective in epidemic situations where rapid reduction in insect pest numbers is required. As such, it can be used in urban dengue control campaigns.
  • Effective space-spraying is generally dependent upon the following specific principles. Target insects are usually flying through the spray cloud (or are sometimes impacted whilst resting on exposed surfaces). The efficiency of contact between the spray droplets and target insects is therefore crucial.
  • a sprayable composition may contain an amount of comprising a combination of one or more CRIPs set for the in Table A, i.e., A1-A68, ranging from about 0.005 wt% to about 99 wt%.
  • a sprayable composition may contain an amount of comprising a combination of one or more Insecticidal Agents set forth in Table B, i.e., B1- B479, ranging from about 0.005 wt% to about 99 wt%.
  • the active compositions of the present invention comprising a combination of (1) one or more CRIPs set for the in Table A, i.e., A1-A68, (2) one or more Insecticidal Agents set forth in Table B, i.e., B1-B479, and (3) an excipient, may be made available in a spray product as an aerosol-based application, including aerosolized foam applications. Pressurized cans are the typical vehicle for the formation of aerosols.
  • An aerosol propellant that is compatible with the CRIP, CRIP-insecticidal protein, or Insecticidal Agent used.
  • a liquefied-gas type propellant is used.
  • Suitable propellants include compressed air, carbon dioxide, butane and nitrogen.
  • the concentration of the propellant in the active compound composition is from about 5 percent to about 40 percent by weight of the pyridine composition, preferably from about 15 percent to about 30 percent by weight of the composition comprising a combination of (1) one or more CRIPs set for the in Table A, i.e., A1-A68, (2) one or more Insecticidal Agents set forth in Table B, i.e., B1-B479, and (3) an excipient, and an excipient.
  • formulations consisting of a TVP, a TVP-insecticidal protein, or a pharmaceutically acceptable salt thereof can also include one or more foaming agents.
  • Foaming agents that can be used include sodium laureth sulfate, cocamide DEA, and cocamidopropyl betaine.
  • the sodium laureth sulfate, cocamide DEA and cocamidopropyl are used in combination.
  • the concentration of the foaming agent(s) in the active compound composition is from about 10 percent to about 25 percent by weight, more preferably 15 percent to 20 percent by weight of the composition.
  • a sprayable composition may contain an amount of comprising a combination of one or more CRIPs set for the in Table A, i.e., A1-A68, ranging from about 0.005 wt% to about 99 wt%.
  • a sprayable composition may contain an amount of comprising a combination of one or more Insecticidal Agents set forth in Table B, i.e., B1- B479, ranging from about 0.005 wt% to about 99 wt%.
  • Burning formulations [00947]
  • a dwelling area may also be treated with an active CRIP, CRIP-insecticidal protein, or Insecticidal Agent composition by using a burning formulation, such as a candle, a smoke coil or a piece of incense containing the composition.
  • the composition may be formulated into household products such as “heated” air fresheners in which insecticidal compositions are released upon heating, e.g., electrically, or by burning.
  • the active compound compositions of the present invention comprising a combination of (1) one or more CRIPs set for the in Table A, i.e., A1-A68, (2) one or more of Insecticidal Agents set forth in Table B, i.e., B1-B479, and (3) an excipient, may be made available in a spray product as an aerosol, a mosquito coil, and/or a vaporizer or fogger.
  • a sprayable composition may contain an amount of comprising a combination of one or more CRIPs set for the in Table A, i.e., A1-A68, ranging from about 0.005 wt% to about 99 wt%.
  • a sprayable composition may contain an amount of comprising a combination of one or more Insecticidal Agents set forth in Table B, i.e., B1- B479, ranging from about 0.005 wt% to about 99 wt%.
  • fabrics and garments may be made containing a pesticidal effective composition comprising a combination of (1) one or more CRIPs set for the in Table A, i.e., A1-A68, (2) one or more Insecticidal Agents set forth in Table B, i.e., B1-B479, and (3) an excipient,.
  • a pesticidal effective composition comprising a combination of (1) one or more CRIPs set for the in Table A, i.e., A1-A68, (2) one or more Insecticidal Agents set forth in Table B, i.e., B1-B479, and (3) an excipient,.
  • the concentration of the CRIP, CRIP- insecticidal protein, or Insecticidal Agent in the polymeric material, fiber, yarn, weave, net, or substrate described herein can be varied within a relatively wide concentration range from, for example, 0.05 to 15 percent by weight, preferably 0.2 to 10 percent by weight, more preferably 0.4 to 8 percent by weight, especially 0.5 to 5, such as 1 to 3, percent by weight.
  • the concentration of the composition comprising a combination of (1) one or more CRIPs set for the in Table A, i.e., A1-A68, (2) one or more Insecticidal Agents set forth in Table B, i.e., B1-B479, and (3) an excipient, (whether for treating surfaces or for coating a fiber, yarn, net, weave) can be varied within a relatively wide concentration range from, for example 0.1 to 70 percent by weight, such as 0.5 to 50 percent by weight, preferably 1 to 40 percent by weight, more preferably 5 to 30 percent by weight, especially 10 to 20 percent by weight.
  • the concentration of the CRIP, CRIP-insecticidal protein, or Insecticidal Agent may be chosen according to the field of application such that the requirements concerning knockdown efficacy, durability and toxicity are met. Adapting the properties of the material can also be accomplished and so custom-tailored textile fabrics are obtainable in this way.
  • an effective amount of a CRIP or Insecticidal Agent can depend on the specific use pattern, the insect pest against which control is most desired and the environment in which the CRIP, CRIP-insecticidal protein, or Insecticidal Agent will be used. Therefore, an effective amount of a CRIP or Insecticidal Agent is sufficient that control of an insect pest is achieved.
  • a sprayable composition may contain an amount of comprising a combination of one or more CRIPs set for the in Table A, i.e., A1-A68, ranging from about 0.005 wt% to about 99 wt%.
  • a sprayable composition may contain an amount of comprising a combination of one or more Insecticidal Agents set forth in Table B, i.e., B1- B479, ranging from about 0.005 wt% to about 99 wt%.
  • compositions or formulations comprising a combination of (1) one or more CRIPs set for the in Table A, i.e., A1-A68, (2) one or more of Insecticidal Agents set forth in Table B, i.e., B1-B479, and (3) an excipient, for coating walls, floors and ceilings inside of buildings, and for coating a substrate or non-living material.
  • compositions comprising a combination of (1) one or more CRIPs set for the in Table A, i.e., A1-A68, (2) one or more Insecticidal Agents set forth in Table B, i.e., B1-B479, and (3) an excipient, can be prepared using known techniques for the purpose in mind.
  • Preparations of compositions comprising a combination of (1) one or more CRIPs set for the in Table A, i.e., A1-A68, (2) one or more of Insecticidal Agents set forth in Table B, i.e., B1-B479, and (3) an excipient could be so formulated to also contain a binder to facilitate the binding of the compound to the surface or other substrate.
  • Agents useful for binding are known in the art and tend to be polymeric in form.
  • the type of binder suitable for a compositions to be applied to a wall surface having particular porosities and/or binding characteristics would be different compared to a fiber, yarn, weave or net—thus, a skilled person, based on known teachings, would select a suitable binder based on the desired surface and/or substrate.
  • Typical binders are poly vinyl alcohol, modified starch, poly vinyl acrylate, polyacrylic, polyvinyl acetate co polymer, polyurethane, and modified vegetable oils.
  • Suitable binders can include latex dispersions derived from a wide variety of polymers and co-polymers and combinations thereof.
  • Suitable latexes for use as binders in the inventive compositions comprise polymers and copolymers of styrene, alkyl styrenes, isoprene, butadiene, acrylonitrile lower alkyl acrylates, vinyl chloride, vinylidene chloride, vinyl esters of lower carboxylic acids and alpha, beta-ethylenically unsaturated carboxylic acids, including polymers containing three or more different monomer species copolymerized therein, as well as post-dispersed suspensions of silicones or polyurethanes. Also suitable may be a polytetrafluoroethylene (PTFE) polymer for binding the active ingredient to other surfaces.
  • PTFE polytetrafluoroethylene
  • a sprayable composition may contain an amount of comprising a combination of one or more CRIPs set for the in Table A, i.e., A1-A68, ranging from about 0.005 wt% to about 99 wt%.
  • a sprayable composition may contain an amount of comprising a combination of one or more Insecticidal Agents set forth in Table B, i.e., B1- B479, ranging from about 0.005 wt% to about 99 wt%.
  • an insecticidal formulation according to the present disclosure may comprising a combination of (1) one or more CRIPs set for the in Table A, i.e., A1-A68, (2) one or more of Insecticidal Agents set forth in Table B, i.e., B1- B479, and (3) an excipient, e.g., diluent or carrier (e.g., such as water), a polymeric binder, and/or additional components such as a dispersing agent, a polymerizing agent, an emulsifying agent, a thickener, an alcohol, a fragrance, or any other inert excipients used in the preparation of sprayable insecticides known in the art.
  • an excipient e.g., diluent or carrier (e.g., such as water), a polymeric binder, and/or additional components such as a dispersing agent, a polymerizing agent, an emulsifying agent, a thickener, an alcohol,
  • a composition comprising a combination of (1) one or more CRIPs set for the in Table A, i.e., A1-A68, (2) one or more Insecticidal Agents set forth in Table B, i.e., B1-B479, and (3) an excipient, can be prepared in a number of different forms or formulation types, such as suspensions or capsules suspensions. And a person skilled in the art can prepare the relevant composition based on the properties of the particular CRIP, CRIP-insecticidal protein, or Insecticidal Agent, its uses, and also its application type.
  • the CRIP, CRIP-insecticidal protein, or Insecticidal Agent used in the methods, embodiments, and other aspects of the present disclosure may be encapsulated in a suspension or capsule suspension formulation.
  • An encapsulated CRIP, CRIP-insecticidal protein, or Insecticidal Agent can provide improved wash-fastness, and also a longer period of activity.
  • the formulation can be organic based or aqueous based, preferably aqueous based.
  • a sprayable composition may contain an amount of comprising a combination of one or more CRIPs set for the in Table A, i.e., A1-A68, ranging from about 0.005 wt% to about 99 wt%.
  • a sprayable composition may contain an amount of comprising a combination of one or more Insecticidal Agents set forth in Table B, i.e., B1- B479, ranging from about 0.005 wt% to about 99 wt%.
  • Microencapsulation [00967] Microencapsulation [00968] Microencapsulated CRIP, CRIP-insecticidal protein, or Insecticidal Agent suitable for use in the compositions and methods according to the present disclosure may be prepared with any suitable technique known in the art. For example, various processes for microencapsulating material have been previously developed. These processes can be divided into three categories: physical methods, phase separation, and interfacial reaction. In the physical methods category, microcapsule wall material and core particles are physically brought together and the wall material flows around the core particle to form the microcapsule.
  • microcapsules are formed by emulsifying or dispersing the core material in an immiscible continuous phase in which the wall material is dissolved and caused to physically separate from the continuous phase, such as by coacervation, and deposit around the core particles.
  • interfacial reaction category microcapsules are formed by emulsifying or dispersing the core material in an immiscible continuous phase and then an interfacial polymerization reaction is caused to take place at the surface of the core particles.
  • concentration of the CRIP, CRIP-insecticidal protein, or Insecticidal Agent present in the microcapsules can vary from 0.1 to 60% by weight of the microcapsule.
  • a sprayable composition may contain an amount of comprising a combination of one or more CRIPs set for the in Table A, i.e., A1-A68, ranging from about 0.005 wt% to about 99 wt%.
  • a sprayable composition may contain an amount of comprising a combination of one or more Insecticidal Agents set forth in Table B, i.e., B1- B479, ranging from about 0.005 wt% to about 99 wt%.
  • Kits, formulations, dispersants, and the ingredients thereof [00972]
  • the formulation used in the compositions (comprising a combination of (1) one or more CRIPs set for the in Table A, i.e., A1-A68, (2) one or more Insecticidal Agents set forth in Table B, i.e., B1-B479, and (3) an excipient,), methods, embodiments and other aspects according to the present disclosure, may be formed by mixing all ingredients together with water, and optionally using suitable mixing and/or dispersing aggregates. In general, such a formulation is formed at a temperature of from 10 to 70°C, preferably 15 to 50°C, more preferably 20 to 40°C.
  • a formulation comprising one or more of (A), (B), (C), and/or (D) is possible, wherein it is possible to use: a CRIP and Insecticidal Agent (as pesticide) (A); solid polymer (B); optional additional additives (D); and to disperse them in the aqueous component (C).
  • as pesticide A
  • solid polymer B
  • optional additional additives D
  • disperse them in the aqueous component C
  • a binder is present in a composition of the present invention (comprising a combination of (1) one or more CRIPs set for the in Table A, i.e., A1-A68, (2) one or more Insecticidal Agents set forth in Table B, i.e., B1-B479, and (3) an excipient,)
  • dispersions of the polymeric binder (B) in water as well as aqueous formulations of the CRIP, CRIP-insecticidal protein, or Insecticidal Agent (A) in water which have been separately prepared before.
  • Such separate formulations may contain additional additives for stabilizing (A) and/or (B) in the respective formulations and are commercially available.
  • Such raw formulations and optionally additional water (component (C)) are added.
  • components (C) optionally additional water (component (C)
  • combinations of the abovementioned ingredients based on the foregoing scheme are likewise possible, e.g., using a pre-formed dispersion of (A) and/or (B) and mixing it with solid (A) and/or (B).
  • a dispersion of the polymeric binder (B) may be a pre-manufactured dispersion already made by a chemicals manufacturer.
  • Such dispersions may be made by providing a mixture of about 20 percent of the binder (B) in water, heating the mixture to temperature of 90°C to 100°C and intensively stirring the mixture for several hours. It is possible to manufacture the formulation as a final product so that it can be readily used by the end-user for the process according to the present invention. And, it is of course similarly possible to manufacture a concentrate, which may be diluted by the end-user with additional water (C) to the desired concentration for use.
  • a composition comprising a combination of (1) one or more CRIPs set for the in Table A, i.e., A1-A68, (2) one or more Insecticidal Agents set forth in Table B, i.e., B1-B479, and (3) an excipient,
  • suitable for SSI application or a coating formulation comprising a combination of (1) one or more CRIPs set for the in Table A, i.e., A1-A68, (2) one or more Insecticidal Agents set forth in Table B, i.e., B1-B479, and (3) an excipient,
  • an exemplary solid formulation of a CRIP or Insecticidal Agent is generally milled to a desired particle size, such as the particle size distribution d(0.5) is generally from 3 to 20, preferably 5 to 15, especially 7 to 12, ⁇ m.
  • a kit comprising at least a first component comprising a combination of (1) one or more CRIPs set for the in Table A, i.e., A1-A68, (2) one or more of Insecticidal Agents set forth in Table B, i.e., B1-B479 (A); and a second component comprising at least one polymeric binder (B).
  • Further additives (D) may be a third separate component of the kit, or may be already mixed with components (A) and/or (B).
  • the end-user may prepare the formulation for use by just adding water (C) to the components of the kit and mixing.
  • the components of the kit may also be formulations in water. Of course it is possible to combine an aqueous formulation of one of the components with a dry formulation of the other component(s).
  • the kit can consist of one formulation of comprising a combination of (1) one or more CRIPs set for the in Table A, i.e., A1-A68, (2) one or more of Insecticidal Agents set forth in Table B, i.e., B1-B479, (A) and optionally water (C); and a second, separate formulation of at least one polymeric binder (B), water as component (C) and optionally components (D).
  • concentrations of the components (A), (B), (C) and optionally (D) will be selected by the skilled artisan depending of the technique to be used for coating/treating.
  • the amount of a combination of (1) one or more CRIPs set for the in Table A, i.e., A1-A68, (2) one or more of Insecticidal Agents set forth in Table B, i.e., B1-B479, (A) may be up to 50, preferably 1 to 50, such as 10 to 40, especially 15 to 30, percent by weight, based on weight of the composition.
  • the amount of polymeric binder (B) may be in the range of 0.01 to 30, preferably 0.5 to 15, more preferably 1 to 10, especially 1 to 5, percent by weight, based on weight of the composition.
  • the amount of additional components (D) is from 0.1 to 20, preferably 0.5 to 15, percent by weight, based on weight of the composition.
  • suitable amounts of pigments and/or dyestuffs and/or fragrances are in general 0.01 to 5, preferably 0.1 to 3, more preferably 0.2 to 2, percent by weight, based on weight of the composition.
  • a typical formulation ready for use comprises 0.1 to 40, preferably 1 to 30, percent of components (A), (B), and optionally (D), the residual amount being water (C).
  • a typical concentration of a concentrate to be diluted by the end-user may comprise 5 to 70, preferably 10 to 60, percent of components (A), (B), and optionally (D), the residual amount being water (C).
  • any of the CRIPs set for the in Table A, i.e., A1-A68, or the Insecticidal Agents set forth in Table B, i.e., B1-B479, as described herein; and/or any of the methods regarding the same, can be used to create any of the foregoing sprayable compositions, formulations, and/or kits as described herein.
  • TVP COMPOSITIONS [00980] Vitrification
  • Vitrification describes a process wherein the reaction kinetics of a peptide are slowed down via immobilization of the peptide in a rigid, amorphous glassy sugar matrix: this results in drastically slowing down degradation of the peptide.
  • a CRIP or a CRIP-insecticidal protein e.g., a TVP of the present invention
  • a TVP of the present invention can be vitrified.
  • a TVP of the present invention can be stabilized using the process of vitrification.
  • vitrification can occur via the use of sugar.
  • the sugar can be trehalose.
  • Trehalose is a disaccharide formed by a 1,1-glycosidic bond between two ⁇ - glucose units.
  • he molecular formula for trehalose is C 12 H 22 O 11 ; having a molecular weight of 342.3 g/mol.
  • Trehalose is found in nature as a disaccharide and also as a monomer in some polymers; however, some trehalose isomers exist that are not found in nature.
  • Trehalose has been shown to stabilize proteins and cells against stresses such as heat, freezing, and desiccation.
  • K. Lippert and E. Galinski Appl. Microbiol. Biotechnol., 1992, 37, 61-65; J. K. Kaushik and R. Bhat, J. Biol. Chem., 2003, 278, 26458- 26465; R. P. Baptista, S. Pedersen, G. J. Cabrita, D. E. Otzen, J. M. Cabral and E. P.
  • Trehalose is well known in the art. Trehalose is readily available from commercial sources. For example, D-(+)-Trehalose dihydrate (Product No. T9531); and Trehalose (Product Nos. PHR1344 and 1673715) are available from Sigma Aldrich (Sigma- Aldrich Corp. St. Louis, MO, USA). [00991] Exemplary trehalose molecules are provided herein, having an Chemical Abstracts Service (CAS) Reg. No.99-20-7 (anhydrous); and CAS Reg. No.6138-23-4 (dihydrous). An exemplary trehalose compound of the present disclosure has a PubChem CID No.7427.
  • CAS Chemical Abstracts Service
  • An exemplary trehalose compound of the present disclosure has a PubChem CID No.7427.
  • a formulation comprising an Insecticidal Agent (IA) and a TVP, TVP-insecticidal protein, or a pharmaceutically acceptable salt thereof can be a liquid concentrate, a wettable powder, or a granule formulation.
  • any of the TVPs, TVP-insecticidal proteins, or pharmaceutically acceptable salts thereof, as described herein can be used in the any of the formulations described below, e.g., any of the foregoing TVPs, TVP-insecticidal proteins, or pharmaceutically acceptable salts thereof, can be used in the formulation of: a wettable powder or granule formulation; or a liquid concentrate formulation.
  • a formulation comprises, consists essentially of, or consists of: (1) one or more IAs as described herein (e.g., those enumerated in Table B; (2) a TVP, a TVP-insecticidal protein, or a pharmaceutically acceptable salt thereof; and (3) one or more excipients; wherein the excipients comprise, consist essentially of, or consist of: trehalose; potassium phosphate dibasic (K 2 HPO 4 ); potassium phosphate monobasic (KH 2 PO 4 ); maltodextrin; and BIT.
  • a formulation of the present invention comprises, a concentration of trehalose ranging from about 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%,
  • a formulation of the present invention comprises, a concentration of trehalose ranging from about 0.1% to about 99.9%; from about 1% to about 99.9%; from about 2% to about 99.9%; from about 3% to about 99.9%; from about 4% to about 99.9%; from about 5% to about 99.9%; from about 6% to about 99.9%; from about 7% to about 99.9%; from about 8% to about 99.9%; from about 9% to about 99.9%; from about 10% to about 99.9%; from about 11% to about 99.9%; from about 12% to about 99.9%; from about 13% to about 99.9%; from about 14% to about 99.9%; from about 15% to about 99.9%; from about 16% to about 99.9%; from about 17% to about 99.9%; from about 18% to about 99.9%; from about 19% to about 99.9%; from about 20% to about 99.9%; from about 21% to about 99.9%; from about 22% to about 99.9%; from about 3% to about 99.
  • a formulation of the present invention comprises a concentration of trehalose ranging from about 0.1% to about 99%; from about 0.1% to about 98%; from about 0.1% to about 97%; from about 0.1% to about 96%; from about 0.1% to about 95%; from about 0.1% to about 94%; from about 0.1% to about 93%; from about 0.1% to about 92%; from about 0.1% to about 91%; from about 0.1% to about 90%; from about 0.1% to about 89%; from about 0.1% to about 88%; from about 0.1% to about 87%; from about 0.1% to about 86%; from about 0.1% to about 85%; from about 0.1% to about 84%; from about 0.1% to about 83%; from about 0.1% to about 82%; from about 0.1% to about 81%; from about 0.1% to about 80%; from about 0.1% to about 79%; from about 0.1% to about 78%; from about 0.1% to about 77%; from about 0.1% to about 76%; from about 0.1% to about 75%; from about 0.1%
  • a formulation of the present invention comprises a concentration of potassium phosphate dibasic (K 2 HPO 4 ) ranging from about 0.1% to about 40%; from about 0.5% to about 40%; from about 1% to about 40%; from about 2% to about 40%; from about 3% to about 40%; from about 4% to about 40%; from about 5% to about 40%; from about 6% to about 40%; from about 7% to about 40%; from about 8% to about 40%; from about 9% to about 40%; from about 10% to about 40%; from about 11% to about 40%; from about 12% to about 40%; from about 13% to about 40%; from about 14% to about 40%; from about 15% to about 40%; from about 16% to about 40%; from about 17% to about 40%; from about 18% to about 40%; from about 19% to about 40%; from about 20% to about 40%; from about 21% to about 40%; from about 22% to about 40%; from about 23% to about 40%; from about 24% to about 40%; from about 25% to about 40%; from about 26% to
  • a formulation of the present invention comprises a concentration of potassium phosphate dibasic (K 2 HPO 4 ) ranging from about 0.1% to about 40%; from about 0.1% to about 39%; from about 0.1% to about 38%; from about 0.1% to about 37%; from about 0.1% to about 36%; from about 0.1% to about 35%; from about 0.1% to about 34%; from about 0.1% to about 33%; from about 0.1% to about 32%; from about 0.1% to about 31%; from about 0.1% to about 30%; from about 0.1% to about 29%; from about 0.1% to about 28%; from about 0.1% to about 27%; from about 0.1% to about 26%; from about 0.1% to about 25%; from about 0.1% to about 24%; from about 0.1% to about 23%; from about 0.1% to about 22%; from about 0.1% to about 21%; from about 0.1% to about 20%; from about 0.1% to about 19%; from about 0.1% to about 18%; from about 0.1% to about 17%; from about 0.1%
  • a formulation of the present invention comprises a concentration of potassium phosphate monobasic (KH 2 PO 4 ) ranging from about 0.1% to about 20%; from about 0.5% to about 20%; from about 1% to about 20%; from about 2% to about 20%; from about 3% to about 20%; from about 4% to about 20%; from about 5% to about 20%; from about 6% to about 20%; from about 7% to about 20%; from about 8% to about 20%; from about 9% to about 20%; from about 10% to about 20%; from about 11% to about 20%; from about 12% to about 20%; from about 13% to about 20%; from about 14% to about 20%; from about 15% to about 20%; from about 16% to about 20%; from about 17% to about 20%; from about 18% to about 20%; or from about 19% to about 20%; wt/wt of the total formulation.
  • KH 2 PO 4 potassium phosphate monobasic
  • a formulation of the present invention comprises a concentration of potassium phosphate monobasic (KH 2 PO 4 ) ranging from about 0.1% to about 20%; from about 0.1% to about 19%; from about 0.1% to about 18%; from about 0.1% to about 17%; from about 0.1% to about 16%; from about 0.1% to about 15%; from about 0.1% to about 14%; from about 0.1% to about 13%; from about 0.1% to about 12%; from about 0.1% to about 11%; from about 0.1% to about 10%; from about 0.1% to about 9%; from about 0.1% to about 8%; from about 0.1% to about 7%; from about 0.1% to about 6%; from about 0.1% to about 5%; from about 0.1% to about 4%; from about 0.1% to about 3%; from about 0.1% to about 2%; from about 0.1% to about 1%; or from about 0.1% to about 0.5%, wt/wt of the total formulation.
  • KH 2 PO 4 potassium phosphate monobasic
  • a formulation of the present invention comprises a concentration of maltodextrin ranging from about 0.1% to about 99.9%; from about 1% to about 99.9%; from about 2% to about 99.9%; from about 3% to about 99.9%; from about 4% to about 99.9%; from about 5% to about 99.9%; from about 6% to about 99.9%; from about 7% to about 99.9%; from about 8% to about 99.9%; from about 9% to about 99.9%; from about 10% to about 99.9%; from about 11% to about 99.9%; from about 12% to about 99.9%; from about 13% to about 99.9%; from about 14% to about 99.9%; from about 15% to about 99.9%; from about 16% to about 99.9%; from about 17% to about 99.9%; from about 18% to about 99.9%; from about 19% to about 99.9%; from about 20% to about 99.9%; from about 21% to about 99.9%; from about 22% to about 99.9%; from about 3% to about 99
  • a formulation of the present invention comprises a concentration of maltodextrin ranging from about 0.1% to about 99%; from about 0.1% to about 98%; from about 0.1% to about 97%; from about 0.1% to about 96%; from about 0.1% to about 95%; from about 0.1% to about 94%; from about 0.1% to about 93%; from about 0.1% to about 92%; from about 0.1% to about 91%; from about 0.1% to about 90%; from about 0.1% to about 89%; from about 0.1% to about 88%; from about 0.1% to about 87%; from about 0.1% to about 86%; from about 0.1% to about 85%; from about 0.1% to about 84%; from about 0.1% to about 83%; from about 0.1% to about 82%; from about 0.1% to about 81%; from about 0.1% to about 80%; from about 0.1% to about 79%; from about 0.1% to about 78%; from about 0.1% to about 77%; from about 0.1% to about 76%; from about 0.1% to about 75%; from
  • the maltodextrin can have a dextrose equivalent ranging from about 2% to about 20%; from about 3% to about 20%; from about 4% to about 20%; from about 5% to about 20%; from about 6% to about 20%; from about 7% to about 20%; from about 8% to about 20%; from about 9% to about 20%; from about 10% to about 20%; from about 11% to about 20%; from about 12% to about 20%; from about 13% to about 20%; from about 14% to about 20%; from about 15% to about 20%; from about 16% to about 20%; from about 17% to about 20%; from about 18% to about 20%; or from about 19% to about 20%; wt/wt of the total formulation.
  • the maltodextrin can have a dextrose equivalent ranging from about 2% to about 20%; from about 2% to about 19%; from about 2% to about 18%; from about 2% to about 17%; from about 2% to about 16%; from about 2% to about 15%; from about 2% to about 14%; from about 2% to about 13%; from about 2% to about 12%; from about 2% to about 11%; from about 2% to about 10%; from about 2% to about 9%; from about 2% to about 8%; from about 2% to about 7%; from about 2% to about 6%; from about 2% to about 5%; from about 2% to about 4%; or from about 2% to about 3%; wt/wt of the total formulation.
  • a formulation of the present invention comprises a concentration of benzisothiazolinone (BIT) ranging from about 0.01% to about 1%; from about 0.025% to about 1%; from about 0.05% to about 1%; from about 0.075% to about 1%; from about 0.1% to about 1%; from about 0.125% to about 1%; from about 0.15% to about 1%; from about 0.175% to about 1%; from about 0.2% to about 1%; from about 0.225% to about 1%; from about 0.25% to about 1%; from about 0.275% to about 1%; from about 0.3% to about 1%; from about 0.325% to about 1%; from about 0.35% to about 1%; from about 0.375% to about 1%; from about 0.4% to about 1%; from about 0.425% to about 1%; from about 0.45% to about 1%; from about 0.475% to about 1%; from about 0.5% to about 1%; from about 0.525% to about 1%; from about 0.55%
  • BIT benzisothi
  • a formulation of the present invention comprises a concentration of benzisothiazolinone (BIT) ranging from about 0.01% to about 1%; from about 0.01% to about 0.975%; from about 0.01% to about 0.95%; from about 0.01% to about 0.925%; from about 0.01% to about 0.9%; from about 0.01% to about 0.875%; from about 0.01% to about 0.85%; from about 0.01% to about 0.825%; from about 0.01% to about 0.8%; from about 0.01% to about 0.775%; from about 0.01% to about 0.725%; from about 0.01% to about 0.7%; from about 0.01% to about 0.675%; from about 0.01% to about 0.65%; from about 0.01% to about 0.625%; from about 0.01% to about 0.6%; from about 0.01% to about 0.575%; from about 0.01% to about 0.55%; from about 0.01% to about 0.525%; from about 0.01% to 0.55%; from about 0.01% to about 0.525%; from about 0.01%
  • the BIT can be 1,2-Benzisothiazolin-3-one.
  • An exemplary 1,2-Benzisothiazolin-3-one is provided herein, having a CAS No.2634-33-5.
  • An exemplary description describing how to make 1,2-Benzisothiazolin-3-one is provided in WIPO Publication No. WO2014173716A1, the disclosure of which is incorporated herein by reference in its entirety.
  • 1,2-Benzisothiazolin-3-one is readily available from commercial vendors, e.g., PROXEL® AQ Preservative; 9.25% aqueous solution of 1,2- benzisothiazolin-3-one; available from Lonza Group Ltd.
  • a formulation of the present invention comprises a concentration of lignosulfonate ranging from about 0.1% to about 1%; from about 0.125% to about 1%; from about 0.15% to about 1%; from about 0.175% to about 1%; from about 0.2% to about 1%; from about 0.225% to about 1%; from about 0.25% to about 1%; from about 0.275% to about 1%; from about 0.3% to about 1%; from about 0.325% to about 1%; from about 0.35% to about 1%; from about 0.375% to about 1%; from about 0.4% to about 1%; from about 0.425% to about 1%; from about 0.45% to about 1%; from about 0.475% to about 1%; from about 0.5% to about 1%; from about 0.525% to about 1%; from about 0.55% to about 1%; from about 0.575% to about 1%; from about 0.6% to about 1%; from about 0.6% to about 1%; from about 0.1%
  • a formulation of the present invention comprises a concentration of lignosulfonate from about 0.1% to about 1%; from about 0.1% to about 0.975%; from about 0.1% to about 0.95%; from about 0.1% to about 0.925%; from about 0.1% to about 0.9%; from about 0.1% to about 0.875%; from about 0.1% to about 0.85%; from about 0.1% to about 0.825%; from about 0.1% to about 0.8%; from about 0.1% to about 0.775%; from about 0.1% to about 0.75%; from about 0.1% to about 0.725%; from about 0.1% to about 0.7%; from about 0.1% to about 0.675%; from about 0.1% to about 0.65%; from about 0.1% to about 0.625%; from about 0.1% to about 0.6%; from about 0.1% to about 0.575%; from about 0.1% to about 0.55%; from about 0.1% to about 0.525%; from about 0.1% to about 0.5%; from about 0.1% to about 0.475%; from about 0.1% to about 0.45%; from about 0.1% to about 0.1% to
  • a formulation of the present invention comprises a concentration of gypsum ranging from about 0.1% to about 1%; from about 0.125% to about 1%; from about 0.15% to about 1%; from about 0.175% to about 1%; from about 0.2% to about 1%; from about 0.225% to about 1%; from about 0.25% to about 1%; from about 0.275% to about 1%; from about 0.3% to about 1%; from about 0.325% to about 1%; from about 0.35% to about 1%; from about 0.375% to about 1%; from about 0.4% to about 1%; from about 0.425% to about 1%; from about 0.45% to about 1%; from about 0.475% to about 1%; from about 0.5% to about 1%; from about 0.525% to about 1%; from about 0.55% to about 1%; from about 0.575% to about 1%; from about 0.6% to about 1%; from about 0.625% to about 1%; from about 0.65% to about 1%; from about 0.67
  • a formulation of the present invention comprises a concentration of gypsum ranging from about 0.1% to about 1%; from about 0.1% to about 0.975%; from about 0.1% to about 0.95%; from about 0.1% to about 0.925%; from about 0.1% to about 0.9%; from about 0.1% to about 0.875%; from about 0.1% to about 0.85%; from about 0.1% to about 0.825%; from about 0.1% to about 0.8%; from about 0.1% to about 0.775%; from about 0.1% to about 0.75%; from about 0.1% to about 0.725%; from about 0.1% to about 0.7%; from about 0.1% to about 0.675%; from about 0.1% to about 0.65%; from about 0.1% to about 0.625%; from about 0.1% to about 0.6%; from about 0.1% to about 0.575%; from about 0.1% to about 0.55%; from about 0.1% to about 0.525%; from about 0.1% to about 0.5%; from about 0.1% to about 0.475%; from about 0.1% to about 0.45%; from about 0.1% to
  • a formulation of the present invention comprises a concentration of sorbitol ranging from about 0.5% to about 8%; from about 0.75% to about 8%; from about 1% to about 8%; from about 1.25% to about 8%; from about 1.5% to about 8%; from about 1.75% to about 8%; from about 2% to about 8%; from about 2.25% to about 8%; from about 2.5% to about 8%; from about 2.75% to about 8%; from about 3% to about 8%; from about 3.25% to about 8%; from about 3.5% to about 8%; from about 3.75% to about 8%; from about 4% to about 8%; from about 4.25% to about 8%; from about 4.5% to about 8%; from about 4.75% to about 8%; from about 5% to about 8%; from about 5.25% to about 8%; from about 5.5% to about 8%; from about 5.75% to about 8%; from about 6% to about 8%; from about 6.25% to about
  • a formulation of the present invention comprises a concentration of sorbitol ranging from about 0.5% to about 8%; from about 0.5% to about 7.75%; from about 0.5% to about 7.5%; from about 0.5% to about 7.25%; from about 0.5% to about 7%; from about 0.5% to about 6.75%; from about 0.5% to about 6.5%; from about 0.5% to about 6.25%; from about 0.5% to about 6%; from about 0.5% to about 5.75%; from about 0.5% to about 5.5%; from about 0.5% to about 5.25%; from about 0.5% to about 5%; from about 0.5% to about 4.75%; from about 0.5% to about 4.5%; from about 0.5% to about 4.25%; from about 0.5% to about 4%; from about 0.5% to about 3.75%; from about 0.5% to about 3.5%; from about 0.5% to about 3.5%; from about 0.5% to about 3.25%; from about 0.5% to about 3%; from about 0.5% to about 2.75%; from about 0.5% to about 2.5%; from about 0.5% to about 2.2
  • a formulation of the present invention comprises a concentration of sodium benzoate ranging from about 0.1% to about 1%; from about 0.125% to about 1%; from about 0.15% to about 1%; from about 0.175% to about 1%; from about 0.2% to about 1%; from about 0.225% to about 1%; from about 0.25% to about 1%; from about 0.275% to about 1%; from about 0.3% to about 1%; from about 0.325% to about 1%; from about 0.35% to about 1%; from about 0.375% to about 1%; from about 0.4% to about 1%; from about 0.425% to about 1%; from about 0.45% to about 1%; from about 0.475% to about 1%; from about 0.5% to about 1%; from about 0.525% to about 1%; from about 0.55% to about 1%; from about 0.575% to about 1%; from about 0.6% to about 1%; from about 0.625% to about 1%; from about 0.65% to about 1%; from about 0.675%
  • a formulation of the present invention comprises a concentration of sodium benzoate ranging from about 0.1% to about 1%; from about 0.1% to about 0.975%; from about 0.1% to about 0.95%; from about 0.1% to about 0.925%; from about 0.1% to about 0.9%; from about 0.1% to about 0.875%; from about 0.1% to about 0.85%; from about 0.1% to about 0.825%; from about 0.1% to about 0.8%; from about 0.1% to about 0.775%; from about 0.1% to about 0.75%; from about 0.1% to about 0.725%; from about 0.1% to about 0.7%; from about 0.1% to about 0.675%; from about 0.1% to about 0.65%; from about 0.1% to about 0.625%; from about 0.1% to about 0.6%; from about 0.1% to about 0.575%; from about 0.1% to about 0.55%; from about 0.1% to about 0.525%; from about 0.1% to about 0.5%; from about 0.1% to about 0.475%; from about 0.1% to about 0.45%; from about 0.1% to about
  • a formulation of the present invention comprises a concentration of potassium sorbate ranging from about 0.1% to about 1%; from about 0.125% to about 1%; from about 0.15% to about 1%; from about 0.175% to about 1%; from about 0.2% to about 1%; from about 0.225% to about 1%; from about 0.25% to about 1%; from about 0.275% to about 1%; from about 0.3% to about 1%; from about 0.325% to about 1%; from about 0.35% to about 1%; from about 0.375% to about 1%; from about 0.4% to about 1%; from about 0.425% to about 1%; from about 0.45% to about 1%; from about 0.475% to about 1%; from about 0.5% to about 1%; from about 0.525% to about 1%; from about 0.55% to about 1%; from about 0.575% to about 1%; from about 0.6% to about 1%; from about 0.625% to about 1%; from about 0.65% to about 1%; from about 0.675%
  • a formulation of the present invention comprises a concentration of potassium sorbate ranging from about 0.1% to about 1%; from about 0.1% to about 0.975%; from about 0.1% to about 0.95%; from about 0.1% to about 0.925%; from about 0.1% to about 0.9%; from about 0.1% to about 0.875%; from about 0.1% to about 0.85%; from about 0.1% to about 0.825%; from about 0.1% to about 0.8%; from about 0.1% to about 0.775%; from about 0.1% to about 0.75%; from about 0.1% to about 0.725%; from about 0.1% to about 0.7%; from about 0.1% to about 0.675%; from about 0.1% to about 0.65%; from about 0.1% to about 0.625%; from about 0.1% to about 0.6%; from about 0.1% to about 0.575%; from about 0.1% to about 0.55%; from about 0.1% to about 0.525%; from about 0.1% to about 0.5%; from about 0.1% to about 0.475%; from about 0.1% to about 0.45%; from about 0.1% to about
  • a formulation of the present invention comprises a concentration of EDTA ranging from about 0.1% to about 1%; from about 0.125% to about 1%; from about 0.15% to about 1%; from about 0.175% to about 1%; from about 0.2% to about 1%; from about 0.225% to about 1%; from about 0.25% to about 1%; from about 0.275% to about 1%; from about 0.3% to about 1%; from about 0.325% to about 1%; from about 0.35% to about 1%; from about 0.375% to about 1%; from about 0.4% to about 1%; from about 0.425% to about 1%; from about 0.45% to about 1%; from about 0.475% to about 1%; from about 0.5% to about 1%; from about 0.525% to about 1%; from about 0.55% to about 1%; from about 0.575% to about 1%; from about 0.6% to about 1%; from about 0.625% to about 1%; from about 0.65% to about 1%; from about 0.675% to
  • a formulation of the present invention comprises a concentration of EDTA ranging from about 0.1% to about 1%; from about 0.1% to about 0.975%; from about 0.1% to about 0.95%; from about 0.1% to about 0.925%; from about 0.1% to about 0.9%; from about 0.1% to about 0.875%; from about 0.1% to about 0.85%; from about 0.1% to about 0.825%; from about 0.1% to about 0.8%; from about 0.1% to about 0.775%; from about 0.1% to about 0.75%; from about 0.1% to about 0.725%; from about 0.1% to about 0.7%; from about 0.1% to about 0.675%; from about 0.1% to about 0.65%; from about 0.1% to about 0.625%; from about 0.1% to about 0.6%; from about 0.1% to about 0.575%; from about 0.1% to about 0.55%; from about 0.1% to about 0.525%; from about 0.1% to about 0.5%; from about 0.1% to about 0.475%; from about 0.1% to about 0.45%; from about 0.1% to about 0.1% to about 0.
  • a formulation of the present invention can be formulated at a pH ranging from about 5 to about 11; from about 5.5 to about 11; from about 6 to about 11; from about 6.5 to about 11; from about 7 to about 11; from about 7.5 to about 11; from about 8 to about 11; from about 8.5 to about 11; from about 9 to about 11; from about 9.5 to about 11; from about 10 to about 11; or from about 10.5 to about 11.
  • a formulation of the present invention can be formulated at a pH ranging from about 5 to about 11; from about 5 to about 10.5; from about 5 to about 10; from about 5 to about 9.5; from about 5 to about 9; from about 5 to about 8.5; from about 5 to about 8; from about 5 to about 7.5; from about 5 to about 7; from about 5 to about 6.5; from about 5 to about 6; or from about 5 to about 5.5.
  • the formulation can be formulated into a granule form (granular formulation).
  • Methods of generating a granular formulation include: crystallization, precipitation, pan-coating, fluid bed coating, agglomeration (e.g., fluid bed agglomeration), rotary atomization, extrusion, prilling, spheronization, size reduction methods, drum granulation, and/or high shear granulation, and the like.
  • the granular formulation can be generated via agglomeration, e.g., spray-drying agglomeration; rewet agglomeration; fluid bed agglomeration; and the like.
  • the type of agglomeration can be fluid bed agglomeration.
  • the granular formulation can be generated via fluid bed agglomeration.
  • the granular formulation can be generated by spraying the active and inert ingredients onto a blank carrier in a fluid bed.
  • the granular formulation can be generated by spraying the active and inert ingredients (excipients) onto a blank carrier and granulated in pan granulator.
  • the granular formulation can be generated by mixing the active and inert powders (i.e., one or more excipients described herein) and water, and subsequently granulated by passing the ingredients through an extruder.
  • the granular formulation can be generated by mixing the active and inert powders (i.e., one or more excipients described herein) with water, and granulated by roll compaction.
  • ILLUSTRATIVE COMBINATIONS, COMPOSITIONS, AND PRODUCTS [001035] The present disclosure contemplates combinations, compositions, and products, comprising one or more CRIPs and one or more Insecticidal Agents (IAs).
  • IAs Insecticidal Agents
  • Any of the combinations, combinations, products, polypeptides and/or plants, utilizing a CRIP as described herein and an IA as described herein e.g., a mixture of one or more of CRIPs: A1-A68, and one or more of IAs: B1-B479), can be used to control pests, their growth, and/or the damage caused by their actions, especially their damage to plants.
  • compositions comprising a combination of one or more of CRIPs: A1-A68 and one or more of IAs: B1-B479 can include agrochemical compositions.
  • agrochemical compositions can include, but is not limited to, aerosols and/or aerosolized products, e.g., sprays, fumigants, powders, dusts, and/or gases; seed dressings; oral preparations (e.g., insect food, etc.); transgenic organisms expressing and/or producing a CRIP and/or a CRIP ORF (either transiently and/or stably) and a peptide IA, e.g., a plant or an animal.
  • combinations or compositions comprising one or more of CRIPs: A1-A68 and one or more of IAs: B1-B479 can be used concomitantly, or sequentially with other insecticides proteins, and/or pesticides as described herein.
  • a composition or combination can comprise a combination of one or more of CRIPs: A1-A68 and one or more of IAs: B1-B479, and one or more peptides or polypeptides from another organism.
  • a combination or composition can comprise a combination of one or more of CRIPs: A1-A68 and one or more of IAs: B1-B479, and one or more peptides or polypeptides from a spider, a scorpion, a sea anemone, a cone shell, a snake, a lizard, or a jellyfish.
  • the active ingredients of the present disclosure can be applied in the form of compositions and can be applied to the crop area or plant to be treated, simultaneously or in succession, with other compounds.
  • These compounds can be fertilizers, weed killers, cryoprotectants, surfactants, detergents, pesticidal soaps, dormant oils, polymers, and/or time-release or biodegradable carrier formulations that permit long-term dosing of a target area following a single application of the formulation. They can also be selective herbicides, chemical insecticides, virucides, microbicides, amoebicides, pesticides, fungicides, bacteriocides, nematocides, molluscicides or mixtures of several of these preparations, if desired, together with further agriculturally acceptable carriers, surfactants or application-promoting adjuvants customarily employed in the art of formulation.
  • Suitable carriers and adjuvants can be solid or liquid and correspond to the substances ordinarily employed in formulation technology, e.g. natural or regenerated mineral substances, solvents, dispersants, wetting agents, tackifiers, binders or fertilizers. Likewise, the formulations may be prepared into edible “baits” or fashioned into pest “traps” to permit feeding or ingestion by a target pest of the pesticidal formulation.
  • Methods of applying an active ingredient of the present disclosure or an agrochemical composition of the present disclosure that contains a combination of one or more of CRIPs: A1-A68 and one or more of IAs: B1-B479 produced by the methods described herein of the present disclosure include leaf application, seed coating and soil application.
  • the composition may be formulated as a powder, dust, pellet, granule, spray, emulsion, colloid, solution, or such like, and may be prepared by such conventional means as desiccation, lyophilization, homogenization, extraction, filtration, centrifugation, sedimentation, or concentration of a culture of cells comprising the polypeptide.
  • the polypeptide may be present in a concentration of from about 1% to about 99% by weight.
  • compositions containing a combination of one or more of CRIPs: A1-A68 and one or more of IAs: B1-B479 may be prophylactically applied to an environmental area to prevent infestation by a susceptible pest, for example, a lepidopteran and/or coleopteran pest, which may be killed or reduced in numbers in a given area by the methods of the invention.
  • a susceptible pest for example, a lepidopteran and/or coleopteran pest
  • the pest ingests, or comes into contact with, a pesticidally-effective amount of the polypeptide.
  • the pesticide compositions described herein may be made by formulating either the bacterial, yeast, or other cell, crystal and/or spore suspension, or isolated protein component with the desired agriculturally-acceptable carrier.
  • the compositions may be formulated prior to administration in an appropriate means such as lyophilized, freeze-dried, desiccated, or in an aqueous carrier, medium or suitable diluent, such as saline and/or other buffer.
  • the formulated compositions may be in the form of a dust or granular material, or a suspension in oil (vegetable or mineral), or water or oil/water emulsions, or as a wettable powder, or in combination with any other carrier material suitable for agricultural application.
  • Suitable agricultural carriers can be solid or liquid and are well known in the art.
  • the formulations may be mixed with one or more solid or liquid adjuvants and prepared by various means, e.g., by homogeneously mixing, blending and/or grinding the pesticidal composition with suitable adjuvants using conventional formulation techniques. Suitable formulations and application methods are described in U.S. Pat. No.6,468,523, herein incorporated by reference in its entirety.
  • a composition comprising a combination of one or more of CRIPs: A1-A68 and one or more of IAs: B1-B479 may also comprise additional ingredients, for example, herbicides, chemical insecticides, virucides, microbicides, amoebicides, pesticides, fungicides, bacteriocides, nematocides, molluscicides, polypeptides, and/or one or more of the foregoing mixtures thereof.
  • additional ingredients for example, herbicides, chemical insecticides, virucides, microbicides, amoebicides, pesticides, fungicides, bacteriocides, nematocides, molluscicides, polypeptides, and/or one or more of the foregoing mixtures thereof.
  • a combination of the present invention can be included in a formulation, for example, a formulation composed of a polar aprotic solvent, and or water, and or where the polar aprotic solvent is present in an amount of 1-99 wt%, the polar protic solvent is present in an amount of 1-99 wt%, and the water is present in an amount of 0-98 wt%.
  • the polar aprotic solvent formulations are especially effective when they contain MSO.
  • MSO is a methylated seed oil and surfactant blend that uses methyl esters of soya oil in amounts of between about 80 and 85 percent petroleum oil with 15 to 20 percent surfactant.
  • a combination of the present invention comprises two types of components, wherein the first type of component is an Insecticidal Agent (IA), and the second type of components is a Cysteine Rich Insecticidal Peptide (CRIP), wherein neither the IA nor CRIP are part of a fusion protein; and wherein the combination results in a insecticidal effect; wherein the ratio of IA to CRIP is about 10,000:1, 5,000:1, 1,000:1, 500:1, 250:1, 200:1, 100:1, 99:1, 95:5, 90:10, 85:15, 80:20, 75:25, 70:30, 65:35, 60:40, 55:45, 1:1, 45:55, 40:60, 35:65, 30:70, 25:75, 20:80, 15:85, 10:90, 5:95, 1:99, 1:100, 1:200, 1:250, 1:500, 1:1,000, 1:5,000, or 1:10,000.
  • IA Insecticidal Agent
  • CRIP Cysteine Rich Insec
  • a combination or composition comprises an Insecticidal Agent (IA) (e.g., one or more of IAs: B1-B479 in Table B), and one or more polypeptides derived from a sea anemone.
  • IA Insecticidal Agent
  • the sea anemone polypeptides can be isolated from: Actinia equina; Anemonia erythraea; Anemonia sulcata; Anemonia viridis; Anthopleura elegantissima; Anthopleura fuscoviridis; Anthopleura xanthogrammica; Bunodosoma caissarum; Bunodosoma cangicum; Bunodosoma granulifera; Heteractis crispa; Parasicyonis actinostoloides; Radianthus paumotensis; or Stoichactis helianthus.
  • the sea anemone toxin can be Av2; an Av3; or a variant thereof.
  • a combination or composition comprises an Insecticidal Agent (IA) (e.g., one or more of IAs: B1-B479 in Table B), and one or more of the following sea anemone toxins: Toxin AETX-1 (AETX I), Toxin APETx1, Toxin APETx2, Antihypertensive protein BDS-1 (Blood depressing substance I), Antihypertensive protein BDS-2 (Blood depressing substance II), Neurotoxin Bg-2 (Bg II), Neurotoxin Bg-3 (Bg III), Toxin APE 1-1, Toxin APE 1-2, Neurotoxin-1 (Toxin ATX-I), Neurotoxin-1 (Neurotoxin I), Neurotoxin 1 (Toxin RTX-I), Neurot
  • a combination or composition comprises an Insecticidal Agent (IA) (e.g., one or more of IAs: B1-B479 in Table B), and one or more sea anemone polypeptides having an amino acid sequence as set forth in SEQ ID NOs: 371-411.
  • IA Insecticidal Agent
  • a combination or composition comprises an Insecticidal Agent (IA) (e.g., one or more of IAs: B1-B479 in Table B), and one or more polypeptides derived from the sea anemone, Anemonia viridis, which possesses a variety of toxins that it uses to defend itself.
  • Av3 is a type III sea anemone toxin that inhibits the inactivation of voltage-gated sodium (Na + ) channels at receptor site 3, resulting in contractile paralysis.
  • Na + voltage-gated sodium
  • the binding of an Av3 toxin to site 3 results in the inactivated state of the sodium channel to become destabilized, which in turn causes the channel to remain in the open position (see Blumenthal et al., Voltage-gated sodium channel toxins: poisons, probes, and future promise. Cell Biochem Biophys.2003; 38(2):215-38).
  • Av3 shows high selectivity for crustacean and insect sodium channels, and low selectivity for mammalian sodium channels (see Moran et al., Sea anemone toxins affecting voltage-gated sodium channels - molecular and evolutionary features, Toxicon.2009 Dec 15; 54(8): 1089–1101).
  • An exemplary Av3 polypeptide from Anemonia viridis is provided having the amino acid sequence of SEQ ID NO:44.
  • the ratio of AVP to Insecticidal Agent (IA) can be selected from at least about the following ratios: 10,000:1, 5,000:1, 1,000:1, 500:1, 250:1, 200:1, 100:1, 99:1, 95:5, 90:10, 85:15, 80:20, 75:25, 70:30, 65:35, 60:40, 55:45, 1:1, 45:55, 40:60, 35:65, 30:70, 25:75, 20:80, 15:85, 10:90, 5:95, 1:99, 1:100, 1:200, 1:250, 1:500, 1:1,000, 1:5,000, or 1:10,000, or any combination of any two of these values.
  • IA Insecticidal Agent
  • the total concentration of AVP and Insecticidal Agent (IA) (e.g., one or more of IAs: B1-B479 in Table B) in the composition is selected from the following percent concentrations: 0, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99 or 100%, or any range between any two of these values, and the remaining percentage of the composition is comprised of excipients.
  • a combination or composition comprises a Insecticidal Agent (IA) (e.g., one or more of IAs: B1-B479 in Table B) and one or more polypeptides derived from the sea anemone Av3, for example, one or more of the Av3 variant polypeptide (AVP) can have the following amino acid variation from SEQ ID NO:44: an N-terminal amino acid substitution of R1K relative to SEQ ID NO:44, changing the polypeptide sequence from the wild-type “RSCCPCYWGGCPWGQNCYPEGCSGPKV” to “KSCCPCYWGGCPWGQNCYPEGCSGPKV” (SEQ ID NO:45); C-terminal amino acid can be deleted relative to SEQ ID NO:44, changing the polypeptide sequence from the wild- type “RSCCPCYWGGCPWGQNCYPEGCSGPKV” to “RSCCPCYWGGCPWGQNCYPEGCSGPK” (SEQ ID NO:
  • the ratio of AVP to Insecticidal Agent (IA) can be selected from at least about the following ratios: 10,000:1, 5,000:1, 1,000:1, 500:1, 250:1, 200:1, 100:1, 99:1, 95:5, 90:10, 85:15, 80:20, 75:25, 70:30, 65:35, 60:40, 55:45, 1:1, 45:55, 40:60, 35:65, 30:70, 25:75, 20:80, 15:85, 10:90, 5:95, 1:99, 1:100, 1:200, 1:250, 1:500, 1:1,000, 1:5,000, or 1:10,000, or any combination of any two of these values.
  • IA Insecticidal Agent
  • the total concentration of AVP and Insecticidal Agent (IA) (e.g., one or more of IAs: B1-B479 in Table B) in the composition is selected from the following percent concentrations: 0, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99 or 100%, or any range between any two of these values, and the remaining percentage of the composition is comprised of excipients.
  • the method of controlling an insect comprises: applying AVP to an insect; and applying an Insecticidal Agent (IA) (e.g., one or more of IAs: B1-B479 in Table B) to said insect.
  • AVP and the Insecticidal Agent (IA) may be applied to insecticidal-resistant insects (e.g., Bt-resistant insects).
  • the ratio of AVP to Insecticidal Agent (IA) can be selected from at least about the following ratios: 10,000:1, 5,000:1, 1,000:1, 500:1, 250:1, 200:1, 100:1, 99:1, 95:5, 90:10, 85:15, 80:20, 75:25, 70:30, 65:35, 60:40, 55:45, 1:1, 45:55, 40:60, 35:65, 30:70, 25:75, 20:80, 15:85, 10:90, 5:95, 1:99, 1:100, 1:200, 1:250, 1:500, 1:1,000, 1:5,000, or 1:10,000, or any combination of any two of these values.
  • IA Insecticidal Agent
  • the total concentration of AVP and Insecticidal Agent (IA) (e.g., one or more of IAs: B1- B479 in Table B) in the composition is selected from the following percent concentrations: 0, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99 or 100%, or any range between any two of these values, and the remaining percentage of the composition is comprised of excipients.
  • IA Insecticidal Agent
  • a combination or composition comprises an Insecticidal Agent (IA) (e.g., one or more of IAs: B1-B479 in Table B), and one or more sea anemone peptides having an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99
  • a combination or composition comprises an Insecticidal Agent (IA) (e.g., one or more of IAs: B1-B479 in Table B) and one or more polypeptides derived, isolated, and/or originating from a spider.
  • IA Insecticidal Agent
  • the spider toxin can be isolated from one of the following species: Phoneutria nigriventer; Allagelena opulenta; Cupiennius salei; Plectreurys tristis; Coremiocnemis valida; Haplopelma huwenum; Agelena orientalis; Allagelena opulenta; Segestria florentina; Apomastus schlingeri; Phoneutria keyserlingi; Macrothele gigas; Macrothele raveni; Missulena bradleyi; Pireneitega luctuosa; Phoneutria reidyi; Illawara wisharti; Eucratoscelus constrictus; Agelenopsis aperta; Hololena curta; Oxyopes lineatus; Brachypelma albiceps; or Brachypelma smithi.
  • the spider toxin can be isolated from Hadronyche versuta (also known as the Blue Mountain funnel web spider), Hadronyche venenata, Atrax robustus, Atrax formidabilis, or Atrax infensus.
  • Hadronyche versuta also known as the Blue Mountain funnel web spider
  • Hadronyche venenata also known as the Blue Mountain funnel web spider
  • Atrax robustus also known as the Blue Mountain funnel web spider
  • Atrax formidabilis also known as the Blue Mountain funnel web spider
  • Atrax infensus also known as the Blue Mountain funnel web spider
  • an Insecticidal Agent (e.g., one or more of IAs: B1-B479 in Table B) can be combined with one or more of the following one of the following spider toxins: U13-ctenitoxin-Pn1a, U13-ctenitoxin-Pn1b,U13-ctenitoxin-Pn1c, U1-agatoxin-Aop1a, U1-ctenitoxin-Cs1a, U1-nemetoxin-Csp1a, U1-nemetoxin-Csp1b, U1- nemetoxin-Csp1c, U1-plectoxin-Pt1a, U1-plectoxin-Pt1b, U1-plectoxin-Pt1c, U1-plectoxin- Pt1d, U1-plectoxin-Pt1f, U1-theraphotoxin-C
  • an Insecticidal Agent (e.g., one or more of IAs: B1-B479 in Table B) can be combined with one or more spider toxins having an amino acid as set forth in SEQ ID NOs: 192-278, and 281-370.
  • an Insecticidal Agent (e.g., one or more of IAs: B1-B479 in Table B) can be combined with one or more ACTX peptides.
  • an Insecticidal Agent (e.g., one or more of IAs: B1-B479 in Table B) can be combined with one or more of the following ACTX peptides: U- ACTX-Hv1a, U+2-ACTX-Hv1a, rU-ACTX-Hv1a, rU-ACTX-Hv1b, r ⁇ -ACTX-Hv1c, ⁇ - ACTX-Hv1a, and/or ⁇ -ACTX-Hv1a+2.
  • IA Insecticidal Agent
  • Exemplary ACTX peptides include: U-ACTX-Hv1a, having the amino acid sequence (SEQ ID NO: 60); U+2-ACTX-Hv1a, having the amino acid sequence (SEQ ID NO: 61); Omega-ACTX-Hv1a, having the amino acid sequence (SEQ ID NO: 62); Omega- hexatoxin-Ar1d, having the amino acid sequence (SEQ ID NO: 63); and Kappa-hexatoxin-Hv1c, having the amino acid sequence (SEQ ID NO: 64).
  • an Insecticidal Agent (e.g., one or more of IAs: B1-B7, B9-B40, and B42-B479 in Table B) can be combined with one or more of the aforementioned exemplary ACTX peptides.
  • a combination or composition comprises an Insecticidal Agent (IA) (e.g., one or more of IAs: B1-B479 in Table B), and one or more U- ACTX peptides, Omega-ACTX peptides, and/or Kappa-ACTX peptides.
  • the ratio of ACTX peptides to Insecticidal Agent (IA) can be selected from at least about the following ratios: 10,000:1, 5,000:1, 1,000:1, 500:1, 250:1, 200:1, 100:1, 99:1, 95:5, 90:10, 85:15, 80:20, 75:25, 70:30, 65:35, 60:40, 55:45, 1:1, 45:55, 40:60, 35:65, 30:70, 25:75, 20:80, 15:85, 10:90, 5:95, 1:99, 1:100, 1:200, 1:250, 1:500, 1:1,000, 1:5,000, or 1:10,000, or any combination of any two of these values.
  • IA Insecticidal Agent
  • the total concentration of ACTX and Insecticidal Agent (IA) (e.g., one or more of IAs: B1-B479 in Table B) in the composition is selected from the following percent concentrations: 0, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99 or 100%, or any range between any two of these values, and the remaining percentage of the composition is comprised of excipients.
  • the method of controlling an insect comprises: applying an ACTX peptide to an insect; and applying an Insecticidal Agent (IA) (e.g., one or more of IAs: B1-B479 in Table B) to said insect.
  • ACTX peptide and the Insecticidal Agent may be applied to insecticidal-resistant insects (e.g., Bt-resistant insects).
  • the ratio of ACTX peptide to Insecticidal Agent (IA) can be selected from at least about the following ratios: 10,000:1, 5,000:1, 1,000:1, 500:1, 250:1, 200:1, 100:1, 99:1, 95:5, 90:10, 85:15, 80:20, 75:25, 70:30, 65:35, 60:40, 55:45, 1:1, 45:55, 40:60, 35:65, 30:70, 25:75, 20:80, 15:85, 10:90, 5:95, 1:99, 1:100, 1:200, 1:250, 1:500, 1:1,000, 1:5,000, or 1:10,000, or any combination of any two of these values.
  • IA Insecticidal Agent
  • the total concentration of ACTX peptide and Insecticidal Agent (IA) (e.g., one or more of IAs: B1-B479 in Table B) in the composition is selected from the following percent concentrations: 0, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99 or 100%, or any range between any two of these values, and the remaining percentage of the composition is comprised of excipients.
  • a combination or composition comprises an Insecticidal Agent (IA) (e.g., one or more of IAs: B1-B479 in Table B) and one or more spider toxins having an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100%
  • a combination or composition comprises an Insecticidal Agent (IA) (e.g., one or more of IAs: B1-B7, B9-B40, and B42-B479 in Table B) and one or more ACTX peptides having an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical
  • an Insecticidal Agent (e.g., one or more of IAs: B1-B479 in Table B) can be combined with one or more ⁇ -CNTX-Pn1a or ⁇ -CNTX- Pn1a toxins.
  • the ⁇ -CNTX-Pn1a peptide is an insecticidal neurotoxin derived from the Brazilian armed spider, Phoneutria nigriventer.
  • ⁇ -CNTX-Pn1a targets the N-methyl-D- aspartate (NMDA)-subtype of ionotropic glutamate receptor (GRIN), and sodium channels.
  • An exemplary ⁇ -CNTX-Pn1a peptide has an amino acid sequence of (SEQ ID NO: 65).
  • the method of controlling an insect comprises: applying ⁇ -CNTX-Pn1a to an insect; and applying an IA to said insect.
  • the foregoing application can be applied concomitantly and/or sequentially, and either in the same or separate compositions.
  • ⁇ -CNTX-Pn1a and Insecticidal Agent (IA) may be applied to ( ⁇ -CNTX-Pn1a)-resistant insects.
  • ⁇ -CNTX-Pn1a and Insecticidal Agent (IA) may be applied to (Bt toxin)-resistant insects.
  • the ratio of ⁇ - CNTX-Pn1a to IA, on a dry weight basis, can be selected from at least about the following ratios: 10,000:1, 5,000:1, 1,000:1, 500:1, 250:1, 200:1, 100:1, 99:1, 95:5, 90:10, 85:15, 80:20, 75:25, 70:30, 65:35, 60:40, 55:45, 1:1, 45:55, 40:60, 35:65, 30:70, 25:75, 20:80, 15:85, 10:90, 5:95, 1:99, 1:100, 1:200, 1:250, 1:500, 1:1,000, 1:5,000, or 1:10,000, or any combination of any two of these values.
  • the total concentration of ⁇ -CNTX-Pn1a and TVP in the composition is selected from the following percent concentrations: 0, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99 or 100%, or any range between any two of these values, and the remaining percentage of the composition is comprised of excipients.
  • the combination or composition comprises both a ⁇ - CNTX-Pn1a and an Insecticidal Agent (IA) (e.g., one or more of IAs: B1-B479 in Table B).
  • IA Insecticidal Agent
  • the combination or composition can be in the ratio of ⁇ -CNTX-Pn1a to Insecticidal Agent (IA) (e.g., one or more of IAs: B1-B479 in Table B), on a dry weight basis, from about any or all of the following ratios: 10,000:1, 5,000:1, 1,000:1, 500:1, 250:1, 200:1, 100:1, 99:1, 95:5, 90:10, 85:15, 80:20, 75:25, 70:30, 65:35, 60:40, 55:45, 1:1, 45:55, 40:60, 35:65, 30:70, 25:75, 20:80, 15:85, 10:90, 5:95, 1:99, 1:100, 1:200, 1:250, 1:500, 1:1,000, 1:5,000, or 1:10,000, or any combination of any two of these values.
  • IA Insecticidal Agent
  • the composition can have a ratio of ⁇ -CNTX-Pn1a to Insecticidal Agent (IA) (e.g., one or more of IAs: B1-B479 in Table B), on a on a dry weight basis, selected from about the following ratios: 0:50, 45:55, 40:60, 35:65, 30:70, 25:75, 20:80, 15:85, 10:90, 5:95, 1:99, 0.5:99.5, 0.1:99.9 and 0.01:99.99 or any combination of any two of these values.
  • IA Insecticidal Agent
  • a combination or composition comprises an Insecticidal Agent (IA) (e.g., one or more of IAs: B1-B479 in Table B) and one or more ⁇ - CNTX-Pn1a peptides having an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.
  • a combination or composition comprises an Insecticidal Agent (IA) (e.g., one or more of IAs: B1-B479 in Table B) and one or more TVPs comprising an amino acid sequence that is at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% identical to the amino acid sequence according to Formula (I): E-P-D-E-I-C-R-X 1 -X 2 -M-X 3 -N-K-E-F-T-Y-X 4 -S-N-V-C- N-N-C-G-D-Q-V-A-A-C-E-A-E-C-F-X 5 -N-D-V-Y-Z 1 -A-C-H-E-A
  • IA Insecticidal Agent
  • a combination or composition comprises an Insecticidal Agent (IA) (e.g., one or more of IAs: B1-B479 in Table B) and one or more TVPs comprising an amino acid sequence that is at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% identical to the amino acid sequence according to Formula (I): E-P-D-E-I-C-R-X 1 -X 2 -M-X 3 -N-K-E-F-T-Y-X 4 -S-N-V-C- N-N-C-G-D-Q-V-A-A-C-E-A-E-C-F-X 5 -N-D-V-Y-Z 1 -A-C-H-E-A-Q-X 6 -X 7 , wherein the polypeptide comprises at least one amino acid substitution relative to the wild-type sequence of
  • a combination or composition comprises an Insecticidal Agent (IA) (e.g., one or more of IAs: B1-B479 in Table B) and one or more TVPs comprising an amino acid sequence that is at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% identical to the amino acid sequence according to Formula (I): E-P-D-E-I-C-R-X 1 -X 2 -M-X 3 -N-K-E-F-T-Y-X 4 -S-N-V-C- N-N-C-G-D-Q-V-A-A-C-E-A-E-C-F-X 5 -N-D-V-Y-Z 1 -A-C-H-E-A-Q-X 6 -X 7 , wherein the polypeptide comprises at least one amino acid substitution relative to the wild-type sequence of
  • a combination or composition comprises an Insecticidal Agent (IA) (e.g., one or more of IAs: B1-B479 in Table B) and one or more TVPs comprising an amino acid sequence that is at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% identical to the amino acid sequence according to Formula (I): E-P-D-E-I-C-R-X 1 -X 2 -M-X 3 -N-K-E-F-T-Y-X 4 -S-N-V-C- N-N-C-G-D-Q-V-A-A-C-E-A-E-C-F-X 5 -N-D-V-Y-Z 1 -A-C-H-E-A-Q-X 6 -X 7 , wherein the polypeptide comprises at least one amino acid substitution relative to the wild-type sequence of
  • a combination or composition comprises an Insecticidal Agent (IA) (e.g., one or more of IAs: B1-B479 in Table B) and one or more TVPs comprising an amino acid sequence that is at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% identical to the amino acid sequence according to Formula (I): E-P-D-E-I-C-R-X 1 -X 2 -M-X 3 -N-K-E-F-T-Y-X 4 -S-N-V-C- N-N-C-G-D-Q-V-A-A-C-E-A-E-C-F-X 5 -N-D-V-Y-Z 1 -A-C-H-E-A-Q-X 6 -X 7 , wherein the polypeptide comprises at least one amino acid substitution relative to the wild-type sequence of
  • a combination or composition comprises an Insecticidal Agent (IA) (e.g., one or more of IAs: B1-B479 in Table B) and one or more TVPs comprising an amino acid sequence that is at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% identical to the amino acid sequence according to Formula (I): E-P-D-E-I-C-R-X 1 -X 2 -M-X 3 -N-K-E-F-T-Y-X 4 -S-N-V-C- N-N-C-G-D-Q-V-A-A-C-E-A-E-C-F-X 5 -N-D-V-Y-Z 1 -A-C-H-E-A-Q-X 6 -X 7 , wherein the polypeptide comprises at least one amino acid substitution relative to the wild-type sequence of
  • a combination or composition comprises an Insecticidal Agent (IA) (e.g., one or more of IAs: B1-B479 in Table B) and one or more TVPs comprising an amino acid sequence that is at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% identical to the amino acid sequence according to Formula (I): E-P-D-E-I-C-R-X 1 -X 2 -M-X 3 -N-K-E-F-T-Y-X 4 -S-N-V-C- N-N-C-G-D-Q-V-A-A-C-E-A-E-C-F-X 5 -N-D-V-Y-Z 1 -A-C-H-E-A-Q-X 6 -X 7 , wherein the polypeptide comprises at least one amino acid substitution relative to the wild-type sequence of
  • a combination or composition comprises an Insecticidal Agent (IA) (e.g., one or more of IAs: B1-B479 in Table B) and one or more TVPs comprising an amino acid sequence that is at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% identical to the amino acid sequence according to Formula (I): E-P-D-E-I-C-R-X 1 -X 2 -M-X 3 -N-K-E-F-T-Y-X 4 -S-N-V-C- N-N-C-G-D-Q-V-A-A-C-E-A-E-C-F-X 5 -N-D-V-Y-Z 1 -A-C-H-E-A-Q-X 6 -X 7 , wherein the polypeptide comprises at least one amino acid substitution relative to the wild-type sequence of
  • a combination or composition comprises an Insecticidal Agent (IA) (e.g., one or more of IAs: B1-B479 in Table B) and one or more TVPs comprising an amino acid sequence that is at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% identical to the amino acid sequence according to Formula (I): E-P-D-E-I-C-R-X 1 -X 2 -M-X 3 -N-K-E-F-T-Y-X 4 -S-N-V-C- N-N-C-G-D-Q-V-A-A-C-E-A-E-C-F-X 5 -N-D-V-Y-Z 1 -A-C-H-E-A-Q-X 6 -X 7 , wherein the polypeptide comprises at least one amino acid substitution relative to the wild-type sequence of
  • a combination or composition comprises an Insecticidal Agent (IA) (e.g., one or more of IAs: B1-B479 in Table B) and one or more TVPs comprising an amino acid sequence that is at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% identical to the amino acid sequence according to Formula (I): E-P-D-E-I-C-R-X 1 -X 2 -M-X 3 -N-K-E-F-T-Y-X 4 -S-N-V-C- N-N-C-G-D-Q-V-A-A-C-E-A-E-C-F-X 5 -N-D-V-Y-Z 1 -A-C-H-E-A-Q-X 6 -X 7 , wherein the polypeptide comprises at least one amino acid substitution relative to the wild-type sequence of
  • a combination or composition comprises an Insecticidal Agent (IA) (e.g., one or more of IAs: B1-B479 in Table B) and one or more TVPs comprising an amino acid sequence that is at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% identical to the amino acid sequence according to Formula (I): E-P-D-E-I-C-R-X 1 -X 2 -M-X 3 -N-K-E-F-T-Y-X 4 -S-N-V-C- N-N-C-G-D-Q-V-A-A-C-E-A-E-C-F-X 5 -N-D-V-Y-Z 1 -A-C-H-E-A-Q-X 6 -X 7 , wherein the polypeptide comprises at least one amino acid substitution relative to the wild-type sequence of
  • a combination or composition comprises an Insecticidal Agent (IA) (e.g., one or more of IAs: B1-B479 in Table B) and one or more TVPs having an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100%
  • a combination or composition comprises an Insecticidal Agent (IA) (e.g., one or more of IAs: B1-B479 in Table B) and one or more Wild-Type U1-agatoxin-Ta1b peptides having an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical,
  • an Insecticidal Agent (e.g., one or more of IAs: B1-B479 in Table B) can be combined with one or more toxins isolated from a scorpion.
  • a combination or composition comprises an Insecticidal Agent (IA) (e.g., one or more of IAs: B1-B479 in Table B) and one or more scorpion toxins selected from the following toxins: Imperatoxin-A (IpTxa), Potassium channel toxin alpha-KTx 10.2 (Cobatoxin-2), Potassium channel toxin alpha-KTx 11.1 (Parabutoxin-1), Potassium channel toxin alpha-KTx 11.2 (Parabutoxin-2), Potassium channel toxin alpha-KTx 11.3 (Parabutoxin-10), Potassium channel toxin alpha-KTx 12.1 (Butantoxin), Potassium channel toxin alpha-KTx 12.2 (Butantoxin), Potassium channel toxin alpha-KTx 12.3 (Butantoxin-like peptide), Potassium channel toxin alpha-KTx 15.1 (Peptide Aa1),
  • a combination or composition comprises an Insecticidal Agent (IA) (e.g., one or more of IAs: B1-B479 in Table B) and one or more scorpion toxins, wherein the scorpion toxin has an amino acid sequence as set forth in SEQ ID NOs: 88-191.
  • a combination or composition comprises an Insecticidal Agent (IA) (e.g., one or more of IAs: B1-B479 in Table B) and one or more scorpion toxins, wherein the toxin can be an imperatoxin.
  • Imperatoxins are peptide toxins derived from the venom of the African scorpion (Pandinus imperator).
  • a combination or composition comprises an Insecticidal Agent (IA) (e.g., one or more of IAs: B1-B479 in Table B) and one or more imperatoxins.
  • IA Insecticidal Agent
  • a combination or composition comprises an Insecticidal Agent (IA) (e.g., one or more of IAs: B1-B479 in Table B) and one or more imperatoxins, wherein the imperatoxin is Imperatoxin A (IpTx-a), or a variant thereof.
  • the IpTx-a has an amino acid sequence of (SEQ ID NO: 66).
  • the method of controlling an insect comprises: applying IpTx-a to an insect; and applying an Insecticidal Agent (IA) (e.g., one or more of IAs: B1-B479 in Table B) to said insect.
  • IA Insecticidal Agent
  • IA and IA may be applied to (IpTx-a)-resistant insects.
  • IpTx-a and Insecticidal Agent may be applied to (Bt toxin)-resistant insects.
  • the ratio of IpTx-a to IA, on a dry weight basis can be selected from at least about the following ratios: 10,000:1, 5,000:1, 1,000:1, 500:1, 250:1, 200:1, 100:1, 99:1, 95:5, 90:10, 85:15, 80:20, 75:25, 70:30, 65:35, 60:40, 55:45, 1:1, 45:55, 40:60, 35:65, 30:70, 25:75, 20:80, 15:85, 10:90, 5:95, 1:99, 1:100, 1:200, 1:250, 1:500, 1:1,000, 1:5,000, or 1:10,000, or any combination of any two of these values.
  • the total concentration of IpTx-a and Insecticidal Agent (IA) (e.g., one or more of IAs: B1-B479 in Table B) in the composition is selected from the following percent concentrations: 0, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99 or 100%, or any range between any two of these values, and the remaining percentage of the composition is comprised of excipients.
  • the combination or composition comprises both an IpTx-a and an Insecticidal Agent (IA) (e.g., one or more of IAs: B1-B479 in Table B).
  • the combination or composition can be in the ratio of IpTx-a to Insecticidal Agent (IA) (e.g., one or more of IAs: B1-B479 in Table B), on a dry weight basis, from about any or all of the following ratios: 10,000:1, 5,000:1, 1,000:1, 500:1, 250:1, 200:1, 100:1, 99:1, 95:5, 90:10, 85:15, 80:20, 75:25, 70:30, 65:35, 60:40, 55:45, 1:1, 45:55, 40:60, 35:65, 30:70, 25:75, 20:80, 15:85, 10:90, 5:95, 1:99, 1:100, 1:200, 1:250, 1:500, 1:1,000, 1:5,000, or 1:10,000, or any combination of any two of these values.
  • IA Insecticidal Agent
  • the combination or composition can have a ratio of IpTx-a to Insecticidal Agent (IA) (e.g., one or more of IAs: B1-B479 in Table B), on a on a dry weight basis, selected from about the following ratios: 0:50, 45:55, 40:60, 35:65, 30:70, 25:75, 20:80, 15:85, 10:90, 5:95, 1:99, 0.5:99.5, 0.1:99.9 and 0.01:99.99 or any combination of any two of these values.
  • IA IpTx-a to Insecticidal Agent
  • an Insecticidal Agent (e.g., one or more of IAs: B1-B479 in Table B) can be combined with one or more AaIT1 toxins.
  • the protein toxin, AalT1 is a sodium channel site 4 toxin from North African desert scorpion (Androctonus australis).
  • An exemplary AaIT1 toxin is a peptide having the amino acid sequence according to SEQ ID NO: 88 (NCBI accession No. P01497.2).
  • AaIT1 is a site 4 toxin, which forces the insect sodium channel to open by lowering the activation reaction energy barrier.
  • the method of controlling an insect comprises: applying AaIT1to an insect; and applying an Insecticidal Agent (IA) (e.g., one or more of IAs: B1-B479 in Table B) to said insect.
  • IA Insecticidal Agent
  • the foregoing application can be applied concomitantly and/or sequentially, and either in the same or separate compositions.
  • AaIT1 and the Insecticidal Agent (IA) may be applied to (AaIT1)-resistant insects.
  • AaIT1 and the Insecticidal Agent (IA) may be applied to (Bt toxin)-resistant insects.
  • the ratio of AaIT1 to Insecticidal Agent (IA) (e.g., one or more of IAs: B1-B479 in Table B), on a dry weight basis, can be selected from at least about the following ratios: 10,000:1, 5,000:1, 1,000:1, 500:1, 250:1, 200:1, 100:1, 99:1, 95:5, 90:10, 85:15, 80:20, 75:25, 70:30, 65:35, 60:40, 55:45, 1:1, 45:55, 40:60, 35:65, 30:70, 25:75, 20:80, 15:85, 10:90, 5:95, 1:99, 1:100, 1:200, 1:250, 1:500, 1:1,000, 1:5,000, or 1:10,000, or any combination
  • the total concentration of AaIT1 and Insecticidal Agent (IA) in the composition is selected from the following percent concentrations: 0, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99 or 100%, or any range between any two of these values, and the remaining percentage of the composition is comprised of excipients.
  • a combination or composition comprises an Insecticidal Agent (IA) (e.g., one or more of IAs: B1-B479 in Table B) and one or more scorpion peptides or scorpion toxins having an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99% identical, at least 99.
  • Conotoxins are toxins isolated from cone shells; these toxins act by interfering with neuronal communication. Examples of conotoxins include the ⁇ -, ⁇ -, ⁇ -, ⁇ -, and ⁇ - conotoxins. Briefly, the ⁇ -conotoxins (and ⁇ A- & ⁇ -conotoxins) target nicotinic ligand gated channels; ⁇ -conotoxins target voltage-gated calcium channels; ⁇ -conotoxins target the voltage-gated sodium channels; ⁇ -conotoxins target the voltage-gated sodium channel; and ⁇ - conotoxins target the voltage-gated potassium channel.
  • an Insecticidal Agent (e.g., one or more of IAs: B1-B479 in Table B), can be combined with one or more peptides isolated from organisms belonging to the Conus genus.
  • an Insecticidal Agent (e.g., one or more of IAs: B1-B479 in Table B) can be combined with one or more peptides isolated from organisms belonging to the Conus genus, wherein the peptide isolated is a conotoxin.
  • an Insecticidal Agent (e.g., one or more of IAs: B1-B479 in Table B) can be combined with one or more peptides isolated from Conus amadis; Conus catus; Conus ermineus; Conus geographus; Conus gloriamaris; Conus kinoshitai; Conus magus; Conus marmoreus; Conus purpurascens; Conus stercusmuscarum; Conus striatus; Conus textile; or Conus tulipa.
  • IA Insecticidal Agent
  • an Insecticidal Agent (e.g., one or more of IAs: B1-B479 in Table B) can be combined with one or more ⁇ -conotoxin, ⁇ A-conotoxin, ⁇ - conotoxins, ⁇ -conotoxin, ⁇ -conotoxin, ⁇ -conotoxin, or ⁇ -conotoxin.
  • the method of controlling an insect comprises: applying a conotoxin to an insect; and applying an Insecticidal Agent (IA) (e.g., one or more of IAs: B1-B479 in Table B) to said insect.
  • conotoxin and Insecticidal Agent may be applied to (conotoxin)-resistant insects.
  • conotoxin and Insecticidal Agent (IA) may be applied to (Bt toxin)-resistant insects.
  • the ratio of conotoxin to Insecticidal Agent (IA) can be selected from at least about the following ratios: 10,000:1, 5,000:1, 1,000:1, 500:1, 250:1, 200:1, 100:1, 99:1, 95:5, 90:10, 85:15, 80:20, 75:25, 70:30, 65:35, 60:40, 55:45, 1:1, 45:55, 40:60, 35:65, 30:70, 25:75, 20:80, 15:85, 10:90, 5:95, 1:99, 1:100, 1:200, 1:250, 1:500, 1:1,000, 1:5,000, or 1:10,000, or any combination of any two of these values.
  • IA conotoxin to Insecticidal Agent
  • the total concentration of conotoxin and Insecticidal Agent (IA) (e.g., one or more of IAs: B1-B479 in Table B) in the composition is selected from the following percent concentrations: 0, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99 or 100%, or any range between any two of these values, and the remaining percentage of the composition is comprised of excipients.
  • the combination or composition comprises both a conotoxin and an Insecticidal Agent (IA) (e.g., one or more of IAs: B1-B479 in Table B).
  • the combination or composition can be in the ratio of conotoxin to Insecticidal Agent (IA) (e.g., one or more of IAs: B1-B479 in Table B), on a dry weight basis, from about any or all of the following ratios: 10,000:1, 5,000:1, 1,000:1, 500:1, 250:1, 200:1, 100:1, 99:1, 95:5, 90:10, 85:15, 80:20, 75:25, 70:30, 65:35, 60:40, 55:45, 1:1, 45:55, 40:60, 35:65, 30:70, 25:75, 20:80, 15:85, 10:90, 5:95, 1:99, 1:100, 1:200, 1:250, 1:500, 1:1,000, 1:5,000, or 1:10,000, or any combination of any two of these values.
  • IA conotoxin to Insecticidal Agent
  • the combination or composition can have a ratio of conotoxin to Insecticidal Agent (IA) (e.g., one or more of IAs: B1-B479 in Table B), on a on a dry weight basis, selected from about the following ratios: 0:50, 45:55, 40:60, 35:65, 30:70, 25:75, 20:80, 15:85, 10:90, 5:95, 1:99, 0.5:99.5, 0.1:99.9 and 0.01:99.99 or any combination of any two of these values.
  • IA conotoxin to Insecticidal Agent
  • a combination or composition of the present invention comprises one or more CRIPs (e.g. one or more of CRIPs: A1-A68 in Table A) and one or more Insecticidal Agents (IAs), wherein the IA is [001116]
  • a combination or composition of the present invention comprises one or more CRIPs (e.g. one or more of CRIPs: A1-A68 in Table A) and one or more Insecticidal Agents (IAs), wherein the IA is [001117]
  • a combination or composition of the present invention comprises one or more CRIPs (e.g.
  • a combination or composition of the present invention comprises one or more CRIPs (e.g. one or more of CRIPs: A1-A68 in Table A) and one or more Insecticidal Agents (IAs), wherein the IA is an entomopathogenic fungi.
  • a combination or composition of the present invention comprises one or more CRIPs (e.g. one or more of CRIPs: A1-A68 in Table A) and one or more Insecticidal Agents (IAs), wherein the IA is a peptide, protein, or toxin produced from an entomopathogenic fungi.
  • a combination or composition of the present invention comprises one or more CRIPs (e.g.
  • a combination or composition of the present invention comprises one or more CRIPs (e.g. one or more of CRIPs: A1-A68 in Table A) and one or more Insecticidal Agents (IAs), wherein the IA is an Ascomycete fungal toxin.
  • a combination or composition of the present invention comprises one or more CRIPs (e.g. one or more of CRIPs: A1-A68 in Table A) and one or more Insecticidal Agents (IAs), wherein the IA is a Cordycipitaceae family fungal toxin.
  • a combination or composition of the present invention comprises one or more CRIPs (e.g.
  • IAs Insecticidal Agents
  • the IA is a Akanthomyces toxin; a Ascopolyporus toxin; a Beauveria toxin; a Beejasamuha toxin; a Cordyceps toxin; a Coremiopsis toxin; a Engyodontium toxin; a Gibellula toxin; a Hyperdermium toxin; a Insecticola toxin; a Isaria toxin; a Lecanicillium toxin; a Microhilum toxin; a Phytocordyceps toxin; a Pseudogibellula toxin; a Rotiferophthora toxin; a Simplicillium toxin; or a Torrubiella toxin.
  • a combination or composition of the present invention comprises one or more CRIPs (e.g. one or more of CRIPs: A1-A68 in Table A) and one or more Insecticidal Agents (IAs), wherein the IA is a fungi organism or toxin therefrom, selected from the following genera: Beauveria; Metarhizium; Paecilomyces; Lecanicillium; Nomuraea; Isaria; Hirsutella; Sorosporella; Aspergillus; Cordiceps; Entomophthora; Zoophthora; Pandora; Entomophaga; Conidiobolus and Basidiobolus.
  • CRIPs e.g. one or more of CRIPs: A1-A68 in Table A
  • IAs Insecticidal Agents
  • a combination or composition of the present invention comprises one or more CRIPs (e.g. one or more of CRIPs: A1-A68 in Table A) and one or more Insecticidal Agents (IAs), wherein the IA is a Beauveria toxin.
  • a combination or composition of the present invention comprises one or more CRIPs (e.g.
  • IAs Insecticidal Agents
  • the IA is one of the following toxins: a Beauveria alba toxin; a Beauveria amorpha toxin; a Beauveria arenaria toxin; a Beauveria asiatica toxin; a Beauveria australis toxin; a Beauveria bassiana toxin; a Cordyceps bassiana toxin; a Beauveria brongniartii toxin; a Beauveria brumptii toxin; a Beauveria caledonica toxin; a Beauveria chiromensis toxin; a Beauveria coccorum toxin; a Beauveria cretacea toxin; a Beauveria cylindrospora toxin; a Beauveria delacro
  • a combination or composition of the present invention comprises one or more CRIPs (e.g. one or more of CRIPs: A1-A68 in Table A) and one or more Insecticidal Agents (IAs), wherein the IA is a Beauveria bassiana toxin
  • a combination or composition of the present invention comprises one or more CRIPs (e.g. one or more of CRIPs: A1-A68 in Table A) and one or more Insecticidal Agents (IAs), wherein the IA is a beauvericin.
  • a combination or composition of the present invention comprises one or more CRIPs (e.g.
  • a combination or composition of the present invention comprises one or more CRIPs (e.g. one or more of CRIPs: A1-A68 in Table A) and one or more Insecticidal Agents (IAs), wherein the IA is a beauvericin having the chemical formula C 45 H 57 N 3 O 9 .
  • a combination or composition of the present invention comprises one or more CRIPs (e.g. one or more of CRIPs: A1-A68 in Table A) and one or more Insecticidal Agents (IAs), wherein the IA is a “Beauvericin A” toxin having the chemical formula C 46 H 59 N 3 O 9 .
  • a combination or composition of the present invention comprises one or more CRIPs (e.g.
  • a combination or composition of the present invention comprises one or more CRIPs (e.g. one or more of CRIPs: A1-A68 in Table A) and one or more Insecticidal Agents (IAs), wherein the IA is a “Beauvericin B” toxin having the chemical formula C 47 H 61 N 3 O 9 .
  • a combination or composition of the present invention comprises one or more CRIPs (e.g. one or more of CRIPs: A1-A68 in Table A) and one or more Insecticidal Agents (IAs), wherein the IA is a Beauveria bassiana strain ANT-03 spore.
  • a combination or composition of the present invention comprises one or more CRIPs (e.g.
  • a combination or composition of the present invention comprises one or more Insecticidal Agents (IAs), and one or more CRIPs (e.g. one or more of CRIPs: A1-A68 in Table A), wherein the IA is a Cordycipitaceae family fungal toxin.
  • a combination or composition of the present invention comprises one or more Insecticidal Agents (IAs), and one or more CRIPs (e.g.
  • IA is an Akanthomyces toxin; a Ascopolyporus toxin; a Beauveria toxin; a Beejasamuha toxin; a Cordyceps toxin; a Coremiopsis toxin; a Engyodontium toxin; a Gibellula toxin; a Hyperdermium toxin; a Insecticola toxin; a Isaria toxin; a Lecanicillium toxin; a Microhilum toxin; a Phytocordyceps toxin; a Pseudogibellula toxin; a Rotiferophthora toxin; a Simplicillium toxin; or a Torrubiella toxin.
  • the IA is an Akanthomyces toxin; a Ascopolyporus toxin; a Beauveria toxin; a Beejasamuha toxin;
  • a combination or composition of the present invention comprises one or more Insecticidal Agents (IAs), and one or more CRIPs (e.g. one or more of CRIPs: A1-A68 in Table A), wherein the IA is a Beauveria toxin.
  • IAs Insecticidal Agents
  • CRIPs e.g. one or more of CRIPs: A1-A68 in Table A
  • the IA is a Beauveria toxin.
  • Combinations: Lectins and CRIPs [001136]
  • a combination of the present invention can comprise one or more IAs, and one or more CRIPs, wherein the IA is not fused nor operably linked to the CRIP, and wherein the IA is Galanthus nivalis agglutinin (GNA).
  • GAA Galanthus nivalis agglutinin
  • a combination or composition of the present invention comprises one or more Insecticidal Agents (IAs), and one or more CRIPs (e.g. one or more of CRIPs: A1-A68 in Table A), wherein the IA is a lectin.
  • a combination or composition of the present invention comprises one or more Insecticidal Agents (IAs), and one or more CRIPs (e.g. one or more of CRIPs: A1-A68 in Table A), wherein the IA is a lectin, wherein said lectin is not fused nor operably linked to the CRIP.
  • a combination or composition of the present invention comprises one or more Insecticidal Agents (IAs), and one or more CRIPs (e.g. one or more of CRIPs: A1-A68 in Table A), wherein the IA is can be one of the following lectins: Galanthus nivalis agglutinin (GNA); Sambucus nigra lectin (SNA); Maackia amurensis-II (MAL-II); Erythrina cristagalli lectin (ECL); Ricinus communis agglutinin-I (RCA); peanut agglutinin (PNA); wheat germ agglutinin (WGA); Griffonia simplicifolia-II (GSL-II); Con A; Lens culinaris agglutinin (LCA); Mannose-binding lectin (MBL); BanLec; galectins; Phaseolus vulgaris Le
  • IA Insectici
  • a combination or composition of the present invention comprises one or more Insecticidal Agents (IAs), and one or more CRIPs (e.g. one or more of CRIPs: A1-A68 in Table A), wherein the IA is a lectin having an amino acid sequence selected from SEQ ID NOs: 35, 595-615, or a variant thereof.
  • IAs Insecticidal Agents
  • CRIPs e.g. one or more of CRIPs: A1-A68 in Table A
  • a combination or composition of the present invention comprises one or more Insecticidal Agents (IAs), and one or more CRIPs (e.g.
  • CRIPs A1-A68 in Table A
  • the IA is one of the following lectins: Galanthus nivalis agglutinin (GNA) (SEQ ID NO: 35); Sambucus nigra (European elder) lectin (SNA) (SEQ ID NO: 596); Leukoagglutinating lectin from the seeds of Maackia amurensis (MAL) (SEQ ID NO: 597); Erythrina cristagalli lectin (ECL) (SEQ ID NO: 598); Ricinus communis agglutinin-I (RCA) (SEQ ID NO: 599); Peanut agglutinin (PNA) (SEQ ID NO: 600); Agglutinin isolectin 1 (WGA1) (SEQ ID NO: 601); Concanavalin-A (CNA) precursor (SEQ ID NO: 602); Jacalin-like lectin (Chain A)

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MX2022013415A (es) 2022-11-14
WO2021222814A1 (en) 2021-11-04
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CA3181913A1 (en) 2021-11-04
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